Antibody-mediated neutralization of ebola viruses

ABSTRACT

The present disclosure is directed to antibodies binding to and neutralizing ebolavirus and methods for use thereof. The present disclosure is directed to a method of detecting an ebolavirus infection in a subject comprising (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Table 2, or an antibody fragment thereof; and (b) detecting ebolavirus glycoprotein in said sample by binding of said antibody or antibody fragment to antigen in said sample. In still further embodiments, the present disclosure concerns immunodetection kits for use with the iminunodetection methods described above.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/138,522, filed Mar. 26, 2015, the entirecontents of which are hereby incorporated by reference.

This invention was made with government support under grant number1U19AI109711 awarded by the National Institutes of Health, and undergrant number HDTRA1-13-1-0034 awarded by the U.S. Defense ThreatReduction Agency (Department of Defense). The government has certainrights in the invention.

BACKGROUND 1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine,infectious disease, and immunology. More particular, the disclosurerelates to antibodies that neutralize ebolavirus.

2. Background

Ebola viruses are members of the family Filoviridae, which infect humansand non-human primates causing a hemorrhagic fever with mortality ratesup to 90%. As of Jan. 7, 2015, there have been in excess of 20,000confirmed, probable, and suspected cases of Ebola virus disease (EVD) inthe current EBOV outbreak in nine affected countries (Guinea, Liberia,Mali, Nigeria, Senegal, Sierra Leone, Spain, the United Kingdom and theUnited States of America) with more than 8,000 deaths (WHO, 2014b).

There is no licensed treatment or vaccine for filovirus infection.Recently, several studies showed that filovirus glycoprotein(GP)-specific neutralizing antibodies (nAbs) can reduce mortalityfollowing experimental inoculation of animals with a lethal dose of EBOV(Dye et al., 2012; Marzi et al., 2012; Olinger et al., 2012; Qiu et al.,2012; Pettitt et al., 2013; Qiu et al., 2014) or MARV (Dye et al.,2012). The primary target of these neutralizing mAbs, the filovirussurface GP, is a trimer composed of three heavily glycosylated GP1-GP2heterodimers. The GP1 subunit can be divided further into base, head,glycan cap and mucin-like domains (Lee et al., 2008). During viralentry, the mucin-like domain and glycan cap mediate binding to multiplehost attachment factors present on the cell membrane. After the virusenters the host cell by macropinocytosis (Nanbo et al., 2010; Saeed etal., 2010), the GP is cleaved by host proteases that removeapproximately 80% of the mass of the GP1 subunit, including themucin-like domain and glycan cap (Chandran et al., 2005; Dube et al.,2009). After cleavage of GP in the endosome, the receptor-binding siteson GP become exposed, and the GP1 head then is able to bind to itsreceptor, Niemann-Pick C1 (NPC 1) protein (Carette et al., 2011;Chandran et al., 2005; Côté et al., 2011). Subsequent conformationalchanges in GP facilitate fusion between viral and endosomal membranes.

The dense clustering of glycans on the glycan cap and mucin-like domainlikely shield much of the surface of EBOV GP from humoral immunesurveillance, leaving only a few sites on the EBOV GP protein where nAbscould bind without interference by glycans (Cook and Lee, 2013). Most ofour knowledge about humoral response against filovirus infections hascome from studies of murine Abs that recognize EBOV GP. From thosestudies, the inventors learned that mouse neutralizing Abspreferentially target peptides exposed in upper, heavily glycosylateddomains or lower areas (the GP1 base) where rearrangements occur thatdrive fusion of viral and host membranes (Saphire, 2013). Abs have notbeen identified that target protein features of the membrane proximalexternal region (MPER) subdomain, which likely rearranges during fusion.Ab KZ52, the only reported human EBOV GP-specific mAb, was obtained froma phage display library that was constructed from bone marrow RNAobtained from a survivor (Maruyama et al., 1999). KZ52 binds a site atthe base of the GP and neutralizes EBOV, most likely by inhibiting theconformational changes required for fusion of viral and endosomalmembranes (Lee et al., 2008). Some murine Abs also have been reported tobind to the base region of Ebola virus GPs (Dias et al., 2011, Murin etal., 2014).

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of detecting an ebolavirus infection in a subject comprising (a)contacting a sample from said subject with an antibody or antibodyfragment having clone-paired heavy and light chain CDR sequences fromTable 2, respectively, or an antibody or antibody fragment thereof asset forth in any figure or Table herein; and (b) detecting ebolavirusglycoprotein in said sample by binding of said antibody or antibodyfragment to antigen in said sample. The sample may be a body fluid, suchas blood, sputum, tears, saliva, mucous or serum, semen urine or feces.Detection may comprise ELISA, RIA, FACS or Western blot. The method mayfurther comprise performing steps (a) and (b) a second time anddetermining a change in the glycoprotein levels as compared to the firstassay.

The antibody or antibody fragment may be characterized by clone-pairedvariable sequences as set forth in Table 3, or light and heavy chainvariable sequences having 70%, 80%, 90% or 95% identity to clone-pairedvariable sequences as set forth in Table 3. The antibody or antibodyfragment may be encoded by light and heavy chain variable sequenceshaving 70%, 80%, 90% or 95% identity to clone-paired variable sequencesas set forth in Table 4. The antibody fragment may be a recombinant ScFv(single chain fragment variable) antibody, Fab fragment, F(ab′)₂fragment, or Fv fragment, or incorporated as scFv or Fab in a diabody.

In another embodiment, there is provided a method of treating a subjectinfected with Ebolavirus, or reducing the likelihood of infection of asubject at risk of contracting Ebolavirus, comprising delivering to saidsubject an antibody or antibody fragment having clone-paired heavy andlight chain CDR sequences from Table 2, respectively, or an antibody orantibody fragment thereof as set forth in any figure or Table herein.The antibody or antibody fragment may be characterized by clone-pairedvariable sequences as set forth in Table 3, or light and heavy chainvariable sequences having 70%, 80%, 90% or 95% identity to clone-pairedvariable sequences as set forth in Table 3. The antibody or antibodyfragment may be encoded by light and heavy chain variable sequenceshaving 70%, 80%, 90% or 95% identity to clone-paired variable sequencesas set forth in Table 4. The antibody fragment may be a recombinant ScFv(single chain fragment variable) antibody, Fab fragment, F(ab′)₂fragment, or Fv fragment, or incorporated as scFv or Fab in a diabody.The antibody may be a chimeric antibody, or is a bispecific antibodythat targets an Ebolavirus antigen other than glycoprotein. The antibodyor fragment thereof may be a bispecific antibody or fragment thereofthat (a) targets a structural feature of an Ebola virus particle, and(b) targets receptor binding domain of Ebola virus. The structuralfeature may be an Ebola virus glycoprotein domain other than thereceptor binding domain. The structural feature may be an Ebola virusvirion structure other than the glycoprotein. The virion structure is alipid, carbohydrate or protein. The antibody or fragment thereof may bea bispecific antibody that (a) targets a structural feature of an Ebolavirus particle and (b) targets a host cell surface structure cells thatis trafficked to endosomes. The host cell surface structure is a virusreceptor (the cholesterol transporter Niemann-Pick C1) or glycan. Theantibody may be administered prior to infection or after infection.Delivering may comprise antibody or antibody fragment administration, orgenetic delivery with an RNA or DNA sequence or vector encoding theantibody or antibody fragment.

In still another embodiment, there is provided a monoclonal antibody,wherein the antibody or antibody fragment thereof is characterized ashaving clone-paired heavy and light chain CDR sequences from Table 2,respectively, or is an antibody or antibody fragment thereof as setforth in any figure or Table herein. The antibody or antibody fragmentmay be characterized by clone-paired variable sequences as set forth inTable 3, or light and heavy chain variable sequences having 70%, 80%,90% or 95% identity to clone-paired variable sequences as set forth inTable 3. The antibody or antibody fragment may be encoded by light andheavy chain variable sequences having 70%, 80%, 90% or 95% identity toclone-paired variable sequences as set forth in Table 4. The antibodyfragment may be a recombinant ScFv (single chain fragment variable)antibody, Fab fragment F(ab′)₂ fragment, or Fv fragment, or incorporatedas scFv or Fab in a diabody. The antibody may be a chimeric antibody, oris a bispecific antibody that targets an Ebolavirus antigen other thanglycoprotein. The antibody or fragment thereof may be a bispecificantibody or fragment thereof that (a) targets a structural feature of anEbola virus particle, and (b) targets receptor binding domain of Ebolavirus. The structural feature may be an Ebola virus glycoprotein domainother than the receptor binding domain. The structural feature may be anEbola virus virion structure other than the glycoprotein. The virionstructure is a lipid, carbohydrate or protein. The antibody or fragmentthereof may be a bispecific antibody that (a) targets a structuralfeature of an Ebola virus particle and (b) targets a host cell surfacestructure cells that is trafficked to endosomes. The host cell surfacestructure is a virus receptor (the cholesterol transporter Niemann-PickC1) or glycan. The antibody may be an IgG. The antibody or antibodyfragment may further comprise a cell penetrating peptide or is anintrabody.

In still a further embodiment, there is provided a hybridoma encoding anantibody or antibody fragment, wherein the antibody or antibody fragmenthas clone-paired heavy and light chain CDR sequences from Table 2,respectively, or is an antibody or antibody fragment thereof as setforth in any figure or Table herein. The antibody or antibody fragmentproduced by the hybridoma may be characterized by clone-paired variablesequences as set forth in Table 3, or light and heavy chain variablesequences having 70%, 80%, 90% or 95% identity to clone-paired variablesequences as set forth in Table 3. The antibody or antibody fragmentproduced by the hybridoma may be encoded by light and heavy chainvariable sequences having 70%, 80%, 90% or 95% identity to clone-pairedvariable sequences as set forth in Table 4. The antibody fragmentproduced by the hybridoma may be a recombinant ScFv (single chainfragment variable) antibody, Fab fragment, F(ab′)₂ fragment, or Fvfragment, or incorporated as scFv or Fab in a diabody. The antibody maybe a chimeric antibody, or is a bispecific antibody that targets anEbolavirus antigen other than glycoprotein. The antibody or fragmentthereof may be a bispecific antibody or fragment thereof that (a)targets a structural feature of an Ebola virus particle, and (b) targetsreceptor binding domain of Ebola virus. The structural feature may be anEbola virus glycoprotein domain other than the receptor binding domain.The structural feature may be an Ebola virus virion structure other thanthe glycoprotein. The virion structure is a lipid, carbohydrate orprotein. The antibody or fragment thereof may be a bispecific antibodythat (a) targets a structural feature of an Ebola virus particle and (b)targets a host cell surface structure cells that is trafficked toendosomes. The host cell surface structure is a virus receptor (thecholesterol transporter Niemann-Pick C1) or glycan. The antibodyproduced by the hybridoma may be an IgG. The antibody or antibodyfragment produced by the hybridoma may further comprise a cellpenetrating peptide or is an intrabody.

Also provided are:

-   -   a human monoclonal antibody or fragment thereof, or a hybridoma        expressing the same, wherein said antibody neutralizes BDBV at 5        ng/ml, and/or neutralizes EBOV at 50 ng/ml;    -   a human monoclonal antibody or fragment thereof, or a hybridoma        expressing the same, wherein said antibody has in IC₅₀ for BDBV        and/or EBOV of 1-1,000 ng/ml; a human monoclonal antibody or        fragment thereof, or a hybridoma expressing the same, wherein        said antibody binds to virus strains of at least two of two        Ebola virus species selected from BDBV, EBOV and SUDV, such as        one that binds to virus strains of all three Ebola virus species        BDBV, EBOV and SUDV, and/or that, neutralizes virus strains of        at least two of Ebola virus species selected from BDBV, EBOV and        SUDV, including one that neutralizes BDBV and EBOV;    -   a human monoclonal antibody antibody fragment or hybridoma        expressing the same, wherein the antibody, antibody fragment or        hybridoma binds to full length Ebolavirus glycoprotein (GP),        Ebolavirus GP with a deleted mucin domain, and secreted        ebolavirus GP;    -   a human monoclonal antibody antibody fragment or hybridoma        expressing the same, wherein the antibody, antibody fragment or        hybridoma binds to full length Ebolavirus glycoprotein (GP) and        Ebolavirus GP with a deleted mucin domain, but not secreted        ebolavirus GP;    -   a human monoclonal antibody antibody fragment or hybridoma        expressing the same, wherein the antibody, antibody fragment or        hybridoma binds to secreted Eebolavirus GP, but not to full        length Ebolavirus glycoprotein (GP) and ebolavirus GP with a        deleted mucin domain;    -   a human monoclonal antibody antibody fragment or hybridoma        expressing the same, wherein the antibody, antibody fragment or        hybridoma binds to the glycan cap domain of Ebolavirus        glycoprotein (GP), or to the heptad repeat region 2 and membrane        proximal external region of the stem of Ebolavirus GP, but does        not bind to the base region of the stem of Ebolavirus GP, such        as where the glycan cap domain is defined as amino acids        227-313, and where the heptad repeat region 2 and membrane        proximal external region is defined as GP amino acids 599-651        (containing the two components 599-632/heptad repeat region 2        and 633-651 [membrane proximal external region), and where the        base region of the stem is defined as the antigenic site at the        surface of the GP1/GP2 interface, recognized by the previously        reported mAbs c4G7, KZ52, c2G4 and 16F6.

Also provided is a vaccine formulation comprising one or more peptidesfrom the membrane proximal external region of Ebolavirus glycoprotein(GP), and a pharmaceutically acceptable buffer, carrier or diluent. Thevaccine formulation may further comprise an adjuvant. The one or morepeptides may comprises a sequence selected from the heptad repeat 2 andMPER region of EBOV, BDBV, or SUDV, such as the EBOV peptidesITDKIDQIIHDFVDK (SEQ ID NO: 25) or TDKIDQIIHDFVDKTL (SEQ ID NO: 26) orthe SUDV peptides ITDKINQIIHDFIDNPL (SEQ ID NO: 27) or TDKINQIIHDFIDNPL(SEQ ID NO: 28) or the BDBV peptide TDKIDQIIHDFIDKPL (SEQ ID NO: 29).The one or more peptides may be from 15-100 residues, from 15-50residues or from 15-25. The one or more peptides may be from 15-50consecutive residues or 15-25 consecutive residues of Ebolavirus. Theone or more peptides may consist of a sequence are selected from theheptad repeat 2 and MPER region of EBOV, BDBV, or SUDV, such as the EBOVpeptides ITDKIDQIIHDFVDK (SEQ ID NO: 25) or TDKIDQIIHDFVDKTL (SEQ ID NO:26) or the SUDV peptides ITDKINQIIHDFIDNPL (SEQ ID NO: 27) orTDKINQIIHDFIDNPL (SEQ ID NO: 28) or the BDBV peptide TDKIDQIIHDFIDKPL(SEQ ID NO: 29).

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Other objects, features and advantages of the present disclosurewill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-D. Cross-reactive B cell responses in filovirus immune donors.Supernatants from EBV-transformed PBMC samples isolated from survivorswere screened in ELISA binding assays using BDBV, EBOV or MARV GPs(FIGS. 1A-C). Results for four BDBV survivors (FIG. 1A), one EBOVsurvivor (FIG. 1B) or one MARV survivor (FIG. 1C) are shown. Height ofthe bars indicates OD_(405 nm) values in ELISA binding to full-lengthextracellular domain of GP of the indicated virus species. Reactivesupernates are color-coded based on the cross-reactivity pattern:species-specific cell lines are highlighted in black, cross-reactivelines to 2 or 3 species are shown in yellow or blue, respectively.Previous work has shown that the amino acid sequence of GP differsbetween BDBV and EBOV by over 34%, and between BDBV and MARV by over72%. (FIG. 1D) Percentages of lines secreting antibodies specific toBDBV, EBOV or MARV GPs, or cross-reactive antibodies to BDBV and EBOV(designated BDBV/EBOV) or BDBV, EBOV and MARV (designatedBDBV/EBOV/MARV) are shown. Increasing intensity of the pink cell fillcolor corresponds to increasing reactivity for indicated virus. See alsoFIG. 8.

FIGS. 2A-D. Cross-neutralizing antibodies from survivors of natural BDBVinfection. (FIG. 2A) Heat map showing the binding of BDBV mAbs to apanel of filovirus GPs. The EC₅₀ value for each GP-mAb combination isshown, with dark red, orange, yellow, or white shading indicating high,intermediate, low, or no detectable binding, respectively. EC₅₀ valuesgreater than 10,000 ng/mL are indicated by the > symbol. NAb names arehighlighted in red. (FIG. 2B) Heat map showing the neutralizationpotency of BDBV GP-specific mAbs against BDBV. The IC₅₀ value for eachvirus-mAb combination is shown. IC₅₀ values greater than 10,000 ng/mLare indicated by the > symbol. Neutralization assays were performed intriplicate. (FIG. 2C) Binding of representative mAbs from six distinctbinding groups to the filovirus GP. (FIG. 2D) Neutralization activity ofrepresentative neutralizing mAbs from three binding groups against BDBV,EBOV or SUDV. Error bars represent the SE of the experiment, performedin triplicate. See also FIGS. 9-11.

FIG. 3. BDBV-neutralizing antibodies target at least two distinctantigenic regions of the GP surface. Data from competition-bindingassays using non-neutralizing mAbs from binding Group 1A (whitebackground) and neutralizing mAbs from binding Groups 1A, 1B, 3A or 3B(pink background). Numbers indicate the percent binding of second mAb inthe presence of the first mAb, compared to binding of un-competed secondmAb. MAbs were judged to compete for the same site if maximum binding ofsecond mAb was reduced to <30% of its un-competed binding (black boxeswith white numbers). MAbs were considered non-competing if maximumbinding of second mAb was >70% of its un-competed binding (white boxeswith red numbers). Grey boxes with black numbers indicate anintermediate phenotype (competition resulted in between 30 and 70% ofun-competed binding). Blue, purple, and green dashed lines indicate whatappear to be major competition groups; the blue and purple groupsoverlap substantially but not completely.

FIGS. 4A-E. BDBV-neutralizing antibodies bind to the glycan cap or baseregion of GP. (FIG. 4A) Shown are negative-stain electron microscopyreference-free 2D class averages of Group 1A antibodies that bind boththe glycan cap of GP and sGP, and Group 1B antibodies that bind theglycan cap of GP but not sGP. BDBV GP or GPΔmuc was used to generatecomplexes. (FIG. 4B) 3D reconstructions of glycan cap binders fromGroups 1A and 1B reveal that these antibodies bind the glycan cap atoverlapping but distinct epitopes. Top (left) and side (right) views ofthe complexes are shown. (FIG. 4C) Reference free 2D class averages ofGroup 1B antibodies (left) reveals that these antibodies bind an epitopebelow the base of GP that is flexible. In the middle image, GP iscolored yellow and each Fab colored green. The right-hand panelillustrates a superimposition of crystal structures of SUDV GPΔmuc (PDB3VEO) and Fabs (PDB 3CSY) to demonstrate how Fabs may bind to GP. (FIG.4D) The composite model delineates the epitopes of the glycan cap mAbsin Group 1A or 1B. Side (above) and top (below) views are shown. (FIG.4E) Docking a crystal structure of SUDV GPΔmuc (PDB 3VEO) (Bale et al.,2012), which contains a more complete model of the glycan cap regiontargeted by Group 1A/B mAbs, reveals how Group 1A/B mAbs target a broadregion in the GP1 centered on the glycan cap, near the beginning of themucin-like domains. Group 1B mAbs that target the base likely bind to aloop near the membrane proximal external region (MPER) that is flexibleand has not yet been resolved at high resolution. TM=transmembraneregion; CT=cytoplasmic tail. See also FIGS. 12A-D.

FIGS. 5A-D. Epitope mapping of Group 3A mAbs using saturationmutagenesis and negative stain electron microscopy. Epitope residues forthree nAbs from Group 3A (BDBV270, BDBV289 and BDBV324) were identifiedas those for which mutation to alanine specifically reduced binding ofthese antibodies (FIGS. 5A-B). GP residue W275 was common to all threenAbs, while L273 was specific for BDBV324, and Y241 was specific forBDBV289. The mutated residues are shown in space filling forms on aribbon diagram of the EBOV GP structure, based on PDB 3CSY. (FIG. 5C)Binding values for nAbs and previously isolated mAbs KZ52, 2G4 and 4G7to library clones with mutations at residues L273, W275 and Y241. The Abreactivities against each mutant EBOV GP clone were calculated relativeto reactivity with wild-type EBOV GP. (FIG. 5D) BDBV289 (brown) binds atthe top of the viral GP near the glycan cap region. Complexes are ofBDBV antibody Fab fragments bound to BDBV GPΔTM with side view (toppanel) or top view (bottom panel). A representative Fab crystalstructure is fit in the Fab density for each reconstruction (from PDBID3CSY). A monomer of the GP trimer crystal structure (PDBID 3CSY) is alsofit in the GP density, with white corresponding to GP1 and black to GP2.Two critical residues for binding by BDBV289 (W275 and Y241, determinedusing saturation mutagenesis) are highlighted in green. See also FIGS.13A-B.

FIGS. 6A-C. Survival and clinical signs of EBOV-inoculated mice treatedwith BDBV mAbs. Groups of 5 mice in each group were injected withindividual mAbs by the intraperitoneal route 1 day after EBOV challenge,using 100 μg of mAb per treatment. Animals treated with denguevirus-specific human mAb 2D22 served as controls. (FIG. 6A) Kaplan-Meiersurvival curves. (FIG. 6B) Body weight. (FIG. 6C) Illness score.

FIGS. 7A-B. Survival and clinical signs of EBOV inoculated guinea pigstreated with BDBV mAbs. Groups of 5 guinea pigs per group were injectedwith individual mAbs by the intraperitoneal route 1 day or 1 and 3 daysafter EBOV challenge, using 5 mg of individual mAb (FIG. 7A) or 5 mg ofthe combination of two mAbs per treatment (FIG. 7B), as indicated.Animals treated with dengue virus-specific human mAb 2D22 served ascontrols. The survival curves are based on morning and eveningobservations. Mortality in the morning is shown in whole day numbers, inthe evening in ½ day values. The body weight and illness scores areshown with one value per day.

FIG. 8. Cross-reactive B cell responses in BDBV immune donors 5 and 6.Related to FIG. 1. Supernatants from EBV-transformed PBMC samplesisolated from survivors were screened in ELISA binding assays usingBDBV, EBOV or MARV GPs. Height of the bars indicates OD_(405 nm) valuesin ELISA binding to full-length extracellular domain of GP of theindicated virus species. Reactive supernates are color-coded based onthe cross-reactivity pattern: species-specific cell lines arehighlighted in black; cross-reactive lines to 2 or 3 species are shownin yellow or blue, respectively.

FIG. 9. Binding patterns of BDBV GP-specific antibodies from BindingGroup 2. (related to FIGS. 2A-D). Antibodies were segregated into sixgroups based on the binding to filovirus GPs. Binding was categorizedbased on the OD₄₀₅ values at the highest antibody concentration tested(E_(max)>0.5) and 50% effective concentration (EC₅₀<10 μg/mL).

FIG. 10. Antibodies from groups 1B, 2B and 3B recognize BDBV GP and BDBVGPΔmuc but not BDBV sGP in ELISA binding assay (related to FIGS. 2A-B).The binding of selected antibodies to BDBV GP, BDBV GPΔmuc and BDBV sGPproteins was tested at a single mAb concentration 10 μg/mL.

FIG. 11. Neutralization activity of BDBV GP-specific nAbs against BDBV(related to FIGS. 2A-D). Red circles represent percent neutralizationrelative to control at different antibody concentrations. Logisticcurves are indicated by solid lines, and 95% confidence intervals areindicated by dashed lines.

FIGS. 12A-E. Raw data and validation of EM models (related to FIGS.4A-E). (FIG. 12A) Raw EM micrograph (far left), 2D reference-free classaverages (middle left), and an FSC curve with resolution indicated (farright) of BDBV41 in complex with BDBV GPΔmuc. (FIG. 12B) As in FIG. 12Abut of BDBV335 in complex with BDBV GPΔmuc. (FIG. 12C) As in FIG. 12A,but of BDBV432 in complex with BDBV GPΔmuc. (FIG. 12D) As in FIG. 12Abut of BDBV353 in complex with BDBV GP. (FIG. 12E) As in FIG. 12A, butof BDBV289 in complex with BDBV GP. Refinement package used to generateeach reconstruction is indicated on the far left. Scale bar indicates200 nm.

FIGS. 13A-B. Generation of escape mutant viruses for BDBV41 (related toFIGS. 5A-E). (FIG. 13A) Neutralization activity of BDBV41 againstwild-type VSV/BDBV-GP (circles, straight curves), VSV/BDBV-GP#7(squares, dashed curves), or VSV/BDBV-GP#15 (triangles, dotted curves)escape mutant viruses. (FIG. 13B) Amino acid changes in BDBV41 escapemutant viruses

FIGS. 14A-C. Cross-reactive neutralizing antibodies from BDBV survivorsbind a unique region on GP surface. (FIG. 14A) Binding of BDBV223,BDBV317 or BDBV340 to BDBV, EBOV, SUDV GP or sGP. (FIG. 14B)Neutralization activity of BDBV223, BDBV317 or BDBV340 against BDBV,EBOV, or SUDV. (FIG. 14C) Data from competition-binding assays usingBDBV223, BDBV317 or BDBV340; antibodies from ZMapp™ cocktail (c2G4, c4G7and 13C6) and previously isolated human antibodies KZ52 and BDBV289.Numbers indicate the percent binding of the second mAb in the presenceof the first mAb, compared to binding of second mAb alone. MAbs werejudged to compete for the same site if maximum binding of the second mAbwas reduced to <30% of its un-competed binding (black boxes with whitenumbers). MAbs were considered non-competing if maximum binding of thesecond mAb was >70% of its un-competed binding (white boxes with rednumbers). Grey boxes with black numbers indicate an intermediatephenotype (between 30 and 70% of un-competed binding).

FIGS. 15A-E. Cross-reactive neutralizing antibodies from BDBV survivorsbind near the membrane proximal region of GP. (FIG. 15A) Representativenegative stain class averages of antibodies that bind GP2 exclusively inthe HR2/MPER region. Complexes are of BDBV Fabs bound to BDBV GPΔmuc.(FIG. 15B) A class average of BDBV GPΔmuc bound to BDBV223 demonstratesthe location of each component, with the core GP colored blue and theFabs in green. (FIG. 15C) A class average of c13C6 Fab:c4G7 Fab bound toEBOV GPΔTM⁷ (with c13C6 in dark blue, c4G7 in yellow and GP core inlight blue). (FIG. 15D) Overlaying a class average of c13C6 Fab:c4G7 Fabbound to EBOV GPΔTM⁷ (with c13C6 in dark blue, c4G7 in yellow and GPcore in light blue) over a class average of BDBV223 Fab bound to BDBVGPΔmuc (with BDBV223 in green and GP core in light blue), demonstratesthat BDBV223 binds significantly lower down on GP, well below theepitope of the c4G7 site of vulnerability at the GP1/GP2 interface.(FIG. 15E) A model of the c13C6 Fab:c4G7 Fab bound to EBOV GPΔTM (EMDBID-6152) is shown with the relative location of BDBV223/317/340 Fabs(segmented c4G7 Fabs from the above map placed in the relative locationon GP as indicated by class averages). Measurements of the distance fromthe bottom of the GP core to the mid-point of the Fab in the classaverages showed a distance of ˜60 Å, which corresponds to the length ofthe HR2 region previously crystalized as post-fusion GP2 (PDBID IEBO).

FIGS. 16A-E. Structural and functional analysis of GP residues importantfor mAb cross-reactivity and neutralization. (FIG. 16A) Sequencealignment of GP2 from BDBV (SEQ ID NO: 344), EBOV (SEQ ID NO: 345) andSUDV (SEQ ID NO: 346). The numbers above the sequence correspond to theamino acid position in GP. Amino acids identical to BDBV are indicatedby dots. Color-coded shapes indicate the position of residues at whichalanine substitutions disrupt mAb binding, as determined byalanine-scanning mutagenesis. BDBV1, BDBV2, EBOV2 or SUDV2 peptidesequences analyzed are indicated by grey, black, blue or purple lines,respectively. (FIG. 16B) Locations of critical residues for BDBV317 andc4G7 binding are displayed on a model of EBOV GP. BDBV317 criticalresidues are highlighted in green and c4G7 critical residues arehighlighted in yellow. (FIG. 16C) Binding of BDBV223, BDBV317 or BDBV340to BDBV1, BDBV2, EBOV2 or SUDV2 peptides. (FIG. 16D) Binding of BDBV223,BDBV317 or BDBV340 to members of a panel of chimeric BDBV peptides(BDBV3=SEQ ID NO: 107).

FIG. 17. Cross-recognition of antibodies from survivors of natural EBOVinfection. Heat map showing the binding of EBOV mAbs to a panel offilovirus GPs. The EC₅₀ value for each GP-mAb combination is shown, withdark red, orange, yellow, or white shading indicating high,intermediate, low, or no detectable binding, respectively. EC₅₀ valuesgreater than 10,000 ng/mL are indicated by the > symbol. NAb names arehighlighted in red.

FIGS. 18A-C. Survival and clinical signs of EBOV-inoculated mice treatedwith EBOV mAbs. Groups of 5 mice in each group were injected withindividual mAbs by the intraperitoneal route 1 day after EBOV challenge,using 100 μg of mAb per treatment. Animals treated with denguevirus-specific human mAb 2D22 served as controls. (FIG. 18A)Kaplan-Meier survival curves. (FIG. 18B) Body weight. (FIG. 18C) Illnessscore. IC₅₀ values in neut test are shown.

FIGS. 19A-C. Survival and clinical signs of EBOV-inoculated mice treatedwith EBOV mAbs. Groups of 5 mice in each group were injected withindividual mAbs by the intraperitoneal route 1 day after EBOV challenge,using 100 μg of mAb per treatment. (FIG. 19A) Kaplan-Meier survivalcurves. (FIG. 19B) Body weight. (FIG. 19C) Illness score. IC₅₀ values inneut test are shown.

FIG. 20. Epitope mapping for representative EBOV mAbs by alaninescanning mutagenesis of EBOV GP.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the work reported here, the inventors isolated a large panel of humanbinding nAbs from B cells of human survivors of severe infection withEbola Bundibugyo species or Ebola Zaire (the latter also called 2014“Makona strain,” or “Guinea strain”, now properly termed Ebolavirusebola) and used these Abs to define the molecular basis of MARVneutralization by human Abs. Remarkably, several of the Abs bound to GPsfrom diverse species of Ebola virus and some neutralized live virusesfrom diverse species. Single-particle EM structures of Ab-GP complexesrevealed that some of the neutralizing Abs bound to EBOV GP near theviral membrane in the membrane proximal external region (MPER). Theantibodies reported here are the first human monoclonal antibodies tothese regions. Additional antibodies were shown to bind at the top ofthe glycoprotein, in the glycan cap region. The inventors isolatedantibodies that recognize distinct patterns of glycoprotein. Some of themonoclonal antibodies bind to full-length and mucin deleted forms ofglycoprotein, while avoiding potential deleterious binding to thesecreted form of glycoprotein. They also found monoclonal antibodiesthat bind to all three forms of glycoprotein, full-length,mucin-deleted, and secreted GP. These and other aspects of thedisclosure are described in detail below.

I. EBOLAVIRUS

The genus Ebolavirus is a virological taxon included in the familyFiloviridae, order Mononegavirales. The members of this genus are calledebolaviruses. The five known virus species are named for the regionwhere each was originally identified: Bundibugyo ebolavirus, Restonebolavirus, Sudan ebolavirus. Taï Forest ebolavirus (originally Côtéd'Ivoire ebolavirus), and Zaire ebolavirus.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon(IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA)nuclear import proteins, preventing their interaction withtyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibitsphospho-STAT1 nuclear import. A biochemical screen now identifiesheterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2)nuclear import as an additional target of VP24. Co-immunoprecipitationstudies demonstrate that hnRNP C1/C2 interacts with multiple KPNA familymembers, including KPNA1. Interaction with hnRNP C1/C2 occurs throughthe same KPNA1 C-terminal region (amino acids 424-457) that binds VP24and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 isdiminished in the presence of VP24, and cells transiently expressingVP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. Thesedata further define the mechanism of hnRNP C1/C2 nuclear import anddemonstrate that the impact of EBOV VP24 on nuclear import extendsbeyond STAT1.

Ebolaviruses were first described after outbreaks of EVD in southernSudan in June 1976 and in Zaire in August 1976. The name Ebolavirus isderived from the Ebola River in Zaire (now the Democratic Republic ofthe Congo), the location of the 1976 outbreak, and the taxonomic suffix-virus (denoting a viral genus). This genus was introduced in 1998 asthe “Ebola-like viruses.” In 2002 the name was changed to Ebolavirus andin 2010, the genus was emended. Ebolaviruses are closely related tomarburgviruses.

Researchers have now found evidence of Ebola infection in three speciesof fruit bats. The bats show no symptoms of the disease, indicating thatthey might be spreading it. Researchers found that bats of threespecies—Hypsignathus monstrosus, Epomops franqueti, and Myonycteristorquata—had either genetic material from the Ebola virus, known as RNAsequences, or evidence of an immune response to the disease. The batsshowed no symptoms themselves. Other hosts are possible as well.

A. Taxonomy

A virus of the family Filoviridae is a member of the genus Ebolavirus ifits genome has several gene overlaps, its fourth gene (GP) encodes fourproteins (sGP, ssGP, Δ-peptide, and GP1.2) using co-transcriptionalediting to express ssGP and GP_(1,2) and proteolytic cleavage to expresssGP and Δ-peptide, peak infectivity of its virions is associated withparticles ≈805 nm in length, its genome differs from that of Marburgvirus by ≥50% and from that of Ebola virus by <50% at the nucleotidelevel, its virions show almost no antigenic cross reactivity withMarburg virions.

The genera Ebolavirus and Marburgvirus were originally classified as thespecies of the now-obsolete Filovirus genus. In March 1998, theVertebrate Virus Subcommittee proposed in the International Committee onTaxonomy of Viruses (ICTV) to change the Filovirus genus to theFiloviridae family with two specific genera: Ebola-like viruses andMarburg-like viruses. This proposal was implemented in Washington, D.C.,as of April 2001 and in Paris as of July 2002. In 2000, another proposalwas made in Washington, D.C., to change the “-like viruses” to “-virus”resulting in today's Ebolavirus and Marburgvirus.

Each species of the genus Ebolavirus has one member virus, and four ofthese cause Ebola virus disease (EVD) in humans, a type of hemorrhagicfever having a very high case fatality rate; the fifth, Reston virus,has caused EVD in other primates. Zaire ebolavirus is the type species(reference or example species) for Ebolavirus, and has the highestmortality rate of the ebolaviruses, and is also responsible for thelargest number of outbreaks of the five known members of the genus,including the 1976 Zaire outbreak and the outbreak with the most deaths(2014). The five characterized species of the Ebolavirus genus are:

Zaire Ebolavirus (ZEBOV).

Also known simply as the Zaire virus, ZEBOV has the highestcase-fatality rate, up to 90% in some epidemics, with an average casefatality rate of approximately 83% over 27 years. There have been moreoutbreaks of Zaire ebolavirus than of any other species. The firstoutbreak took place on 26 Aug. 1976 in Yambuku. Mabalo Lokela, a44-year-old schoolteacher, became the first recorded case. The symptomsresembled malaria, and subsequent patients received quinine.Transmission has been attributed to reuse of unsterilized needles andclose personal contact. The virus is responsible for the 2014 WestAfrica Ebola virus outbreak, with the largest number of deaths to date.

Sudan Ebolavirus (SUDV).

Like ZEBOV, SUDV emerged in 1976; it was at first assumed to beidentical with ZEBOV. SUDV is believed to have broken out first amongstcotton factory workers in Nzara, Sudan (now in South Sudan), in June1976, with the first case reported as a worker exposed to a potentialnatural reservoir. Scientists tested local animals and insects inresponse to this; however, none tested positive for the virus. Thecarrier is still unknown. The lack of barrier nursing (or “bedsideisolation”) facilitated the spread of the disease. The average fatalityrates for SUDV were 54% in 1976, 68% in 1979, and 53% in 2000 and 2001.

Reston Ebolavirus (RESTV).

This virus was discovered during an outbreak of simian hemorrhagic fevervirus (SHFV) in crab-eating macaques from Hazleton Laboratories (nowCovance) in 1989. Since the initial outbreak in Reston, Va., it hassince been found in nonhuman primates in Pennsylvania, Texas, and Siena,Italy. In each case, the affected animals had been imported from afacility in the Philippines, where the virus has also infected pigs.Despite its status as a Level-4 organism and its apparent pathogenicityin monkeys, RESTV did not cause disease in exposed human laboratoryworkers.

Taï Forest ebolavirus (TAFV).

Formerly known as “Côté d'Ivoire ebolavirus,” it was first discoveredamong chimpanzees from the Tai Forest in Côté d'Ivoire, Africa, in 1994.Necropsies showed blood within the heart to be brown; no obvious markswere seen on the organs; and one necropsy displayed lungs filled withblood. Studies of tissues taken from the chimpanzees showed resultssimilar to human cases during the 1976 Ebola outbreaks in Zaire andSudan. As more dead chimpanzees were discovered, many tested positivefor Ebola using molecular techniques. The source of the virus wasbelieved to be the meat of infected western red colobus monkeys(Procoluhbus badius) upon which the chimpanzees preyed. One of thescientists performing the necropsies on the infected chimpanzeescontracted Ebola. She developed symptoms similar to those of denguefever approximately a week after the necropsy, and was transported toSwitzerland for treatment. She was discharged from hospital after twoweeks and had fully recovered six weeks after the infection.

Bundibugyo Ebolavirus (BDBV).

On Nov. 24, 2007, the Uganda Ministry of Health confirmed an outbreak ofEbola in the Bundibugyo District. After confirmation of samples testedby the United States National Reference Laboratories and the CDC, theWorld Health Organization confirmed the presence of the new species. On20 Feb. 2008, the Uganda Ministry officially announced the end of theepidemic in Bundibugyo, with the last infected person discharged on 8Jan. 2008. An epidemiological study conducted by WHO and Uganda Ministryof Health scientists determined there were 116 confirmed and probablecases the new Ebola species, and that the outbreak had a mortality rateof 34% (39 deaths).

B. Ebola Virus Disease

Symptoms of Ebola Virus Disease.

The incubation period from infection with the virus to onset of symptomsis 2 to 21 days. Humans are not infectious until they develop symptoms.First symptoms are the sudden onset of fever fatigue, muscle pain,headache and sore throat. This is followed by vomiting, diarrhea, rash,symptoms of impaired kidney and liver function, and in some cases, bothinternal and external bleeding (e.g., oozing from the gums, blood in thestools). Laboratory findings include low white blood cell and plateletcounts and elevated liver enzymes.

Diagnosis.

It can be difficult to distinguish ebolavirus from other infectiousdiseases such as malaria, typhoid fever and meningitis. Confirmationthat symptoms are caused by ebolavirus infection are made usingantibody-capture ELISA, antigen-capture detection tests, serumneutralization test, RT-PCR assay, electron microscopy, and virusisolation by cell culture. Samples from patients are an extremebiohazard risk; laboratory testing on non-inactivated samples should beconducted under maximum biological containment conditions.

Treatment and Vaccines.

Supportive care-rehydration with oral or intravenous fluids- andtreatment of specific symptoms, improves survival. There is as yet noproven treatment available for ebolavrus. However, a range of potentialtreatments including blood products, immune therapies and drug therapiesare currently being evaluated. No licensed vaccines are available yet,but 2 potential vaccines are undergoing human safety testing.

Prevention and Control.

Good outbreak control relies on applying a package of interventions,namely case management, surveillance and contact tracing, a goodlaboratory service, safe burials and social mobilization. Communityengagement is key to successfully controlling outbreaks. Raisingawareness of risk factors for Ebola infection and protective measuresthat individuals can take is an effective way to reduce humantransmission. Risk reduction messaging should focus on several factors:

-   -   reducing the risk of wildlife-to-human transmission from contact        with infected fruit bats or monkeys/apes and the consumption of        their raw meat;    -   reducing the risk of human-to-human transmission from direct or        close contact with people with Ebola symptoms, particularly with        their bodily fluids;    -   outbreak containment measures including prompt and safe burial        of the dead;    -   identifying people who may have been in contact with someone        infected with Ebola and monitoring the health of contacts for 21        days;    -   the importance of separating the healthy from the sick to        prevent further spread; and    -   the importance of good hygiene and maintaining a clean        environment        In terms of controlling infection in health-care settings,        health-care workers should always take standard precautions when        caring for patients, regardless of their presumed diagnosis.        These include basic hand hygiene, respiratory hygiene, use of        personal protective equipment (to block splashes or other        contact with infected materials), safe injection practices and        safe burial practices. Health-care workers caring for patients        with suspected or confirmed Ebola virus should apply extra        infection control measures to prevent contact with the patient's        blood and body fluids and contaminated surfaces or materials        such as clothing and bedding. When in close contact (within 1        meter) of patients with EBV, health-care workers should wear        face protection (a face shield or a medical mask and goggles), a        clean, non-sterile long-sleeved gown, and gloves (sterile gloves        for some procedures). Laboratory workers are also at risk.        Samples taken from humans and animals for investigation of Ebola        infection should be handled by trained staff and processed in        suitably equipped laboratories.

II. MONOCLONAL ANTIBODIES AND PRODUCTION THEREOF

A. General Methods

It will be understood that monoclonal antibodies binding to Ebolaviruswill have several applications. These include the production ofdiagnostic kits for use in detecting and diagnosing cancer, as well asfor cancer therapies. In these contexts, one may link such antibodies todiagnostic or therapeutic agents, use them as capture agents orcompetitors in competitive assays, or use them individually withoutadditional agents being attached thereto. The antibodies may be mutatedor modified, as discussed further below. Methods for preparing andcharacterizing antibodies are well known in the art (see, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;U.S. Pat. No. 4,196,265).

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Thefirst step for both these methods is immunization of an appropriatehost, or as described below, the identification of subjects who areimmune due to prior natural infection. Antibody-producing cells may beinduced to expand by priming with immunogens. A variety of routes can beused to administer such immunogen (subcutaneous, intramuscular,intradermal, intravenous and intraperitoneal). The production ofpolyclonal antibodies may be monitored by sampling blood of theimmunized animal at various points following immunization. A second,booster injection, also may be given. The process of boosting andtitering is repeated until a suitable titer is achieved. When a desiredlevel of immunogenicity is obtained, the animal can be bled and theserum isolated and stored, and/or the animal can be used to generatemAbs.

Somatic cells with the potential for producing antibodies, specificallyB lymphocytes (B cells), are selected for use in the mAb generatingprotocol. These cells may be obtained from biopsied spleens or lymphnodes, or from circulating blood. The antibody-producing B lymphocytesfrom the immunized animal are then fused with cells of an immortalmyeloma cell, generally one of the same species as the animal that wasimmunized or human or human/mouse chimeric cells. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Any one of a number of myeloma cells may be used, as areknown to those of skill in the art (Goding, pp. 65-66, 1986; Campbell,pp. 75-83, 1984).

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods also is appropriate (Goding, pp.71-74, 1986). Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶; to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, infused cells (particularly the infused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine. Ouabain is added if the B cell source isan Epstein Barr virus (EBV) transformed human B cell line, in order toeliminate EBV transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cellscapable of operating nucleotide salvage pathways are able to survive inHAT medium. The myeloma cells are defective in key enzymes of thesalvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT),and they cannot survive. The B cells can operate this pathway, but theyhave a limited life span in culture and generally die within about twoweeks. Therefore, the only cells that can survive in the selective mediaare those hybrids formed from myeloma and B cells. When the source of Bcells used for fusion is a line of EBV-transformed B cells, as here,ouabain is also used for drug selection of hybrids as EBV-transformed Bcells are susceptible to drug killing, whereas the myeloma partner usedis chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays dot immunobindingassays, and the like. The selected hybridomas are then serially dilutedor single-cell sorted by flow cytometric sorting and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into an animal (e.g., amouse). Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. When human hybridomas are used in this way, it is optimal toinject immunocompromised mice, such as SCID mice, to prevent tumorrejection. The injected animal develops tumors secreting the specificmonoclonal antibody produced by the fused cell hybrid. The body fluidsof the animal, such as serum or ascites fluid, can then be tapped toprovide mAbs in high concentration. The individual cell lines could alsobe cultured in vitro, where the mAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations. Alternatively, human hybridoma cells lines can be usedin vitro to produce immunoglobulins in cell supernatant. The cell linescan be adapted for growth in serum-free medium to optimize the abilityto recover human monoclonal immunoglobulins of high purity.

mAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asFPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the disclosure can be obtained from the purified monoclonalantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, monoclonal antibody fragments encompassed bythe present disclosure can be synthesized using an automated peptidesynthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. For this, RNA can be isolated from thehybridoma line and the antibody genes obtained by RT-PCR and cloned intoan immunoglobulin expression vector. Alternatively, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe cell lines and phagemids expressing appropriate antibodies areselected by panning using viral antigens. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by H and L chaincombination which further increases the chance of finding appropriateantibodies.

Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present disclosure includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobulin preparations; and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in thefirst instance, by their binding specificity, which in this case is forEbolavirus glycoprotein (GP). Those of skill in the art, by assessingthe binding specificity/affinity of a given antibody using techniqueswell known to those of skill in the art, can determine whether suchantibodies fall within the scope of the instant claims. In one aspect,there are provided monoclonal antibodies having clone-paired CDRs fromthe heavy and light chains as illustrated in Table 2. Such antibodiesmay be produced by the clones discussed below in the Examples sectionusing methods described herein.

In a second aspect, the antibodies may be defined by their variablesequence, which include additional “framework” regions. These areprovided in Tables 3-4 that encode or represent full variable regions.Furthermore, the antibodies sequences may vary from these sequences,optionally using methods discussed in greater detail below. For example,nucleic acid sequences may vary from those set out above in that (a) thevariable regions may be segregated away from the constant domains of thelight and heavy chains, (b) the nucleic acids may vary from those setout above while not affecting the residues encoded thereby, (c) thenucleic acids may vary from those set out above by a given percentage,e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% homology, (d) the nucleic acids may vary from those set out above byvirtue of the ability to hybridize under high stringency conditions, asexemplified by low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.15 M NaCl at temperatures of about50° C. to about 70° C., (e) the amino acids may vary from those set outabove by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 990% homology, or (f) the amino acids may varyfrom those set out above by permitting conservative substitutions(discussed below). Each of the foregoing apply to the nucleic acidsequences set forth as Table 3 and the amino acid sequences of Table 4.

C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of theidentified antibodies for a variety of reasons, such as improvedexpression, improved cross-reactivity or diminished off-target binding.The following is a general discussion of relevant techniques forantibody engineering.

Hybridomas may be cultured, then cells lysed, and total RNA extracted.Random hexamers may be used with RT to generate cDNA copies of RNA, andthen PCR performed using a multiplex mixture of PCR primers expected toamplify all human variable gene sequences. PCR product can be clonedinto pGEM-T Easy vector, then sequenced by automated DNA sequencingusing standard vector primers. Assay of binding and neutralization maybe performed using antibodies collected from hybridoma supernatants andpurified by FPLC, using Protein G columns.

Recombinant full length IgG antibodies were generated by subcloningheavy and light chain Fv DNAs from the cloning vector into an IgGplasmid vector, transfected into 293 Freestyle cells or CHO cells, andantibodies were collected an purified from the 293 or CHO cellsupernatant.

The rapid availability of antibody produced in the same host cell andcell culture process as the final cGMP manufacturing process has thepotential to reduce the duration of process development programs. Lonzahas developed a generic method using pooled transfectants grown in CDACFmedium, for the rapid production of small quantities (up to 50 g) ofantibodies in CHO cells. Although slightly slower than a true transientsystem, the advantages include a higher product concentration and use ofthe same host and process as the production cell line. Example of growthand productivity of GS-CHO pools, expressing a model antibody, in adisposable bioreactor: in a disposable bag bioreactor culture (5 Lworking volume) operated in fed-batch mode, a harvest antibodyconcentration of 2 g/L was achieved within 9 weeks of transfection.

Antibody molecules will comprise fragments (such as F(ab′), F(ab′)₂)that are produced, for example, by the proteolytic cleavage of the mAbs,or single-chain immunoglobulins producible, for example, via recombinantmeans. Such antibody derivatives are monovalent. In one embodiment, suchfragments can be combined with one another, or with other antibodyfragments or receptor ligands to form “chimeric” binding molecules.Significantly, such chimeric molecules may contain substituents capableof binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosedantibodies, e.g., an antibody comprising the CDR sequences identical tothose in the disclosed antibodies (e.g., a chimeric, or CDR-graftedantibody). Alternatively, one may wish to make modifications, such asintroducing conservative changes into an antibody molecule. In makingsuch changes, the hydropathic index of amino acids may be considered.The importance of the hydropathic amino acid index in conferringinteractive biologic function on a protein is generally understood inthe art (Kyte and Doolittle, 1982). It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: basic amino acids: arginine (+3.0), lysine (+3.0), andhistidine (−0.5); acidic amino acids: aspartate (+3.0±1), glutamate(+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionicamino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), andthreonine (−0.4), sulfur containing amino acids: cysteine (−1.0) andmethionine (−1.3); hydrophobic, nonaromatic amino acids: valine (−1.5),leucine (−1.8), isoleucine (−1.8), proline (−0.5±1), alanine (−0.5), andglycine (0); hydrophobic, aromatic amino acids: tryptophan (−3.4),phenylalanine (−2.5), and tyrosine (−2.3).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity and produce a biologically orimmunologically modified protein. In such changes, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those that are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine: glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. Bymodifying the Fc region to have a different isotype, differentfunctionalities can be achieved. For example, changing to IgGI canincrease antibody dependent cell cytotoxicity, switching to class A canimprove tissue distribution, and switching to class M can improvevalency.

Modified antibodies may be made by any technique known to those of skillin the art, including expression through standard molecular biologicaltechniques, or the chemical synthesis of polypeptides. Methods forrecombinant expression are addressed elsewhere in this document.

D. Single Chain Antibodies

A Single Chain Variable Fragment (scFv) is a fusion of the variableregions of the heavy and light chains of immunoglobulins, linkedtogether with a short (usually serine, glycine) linker. This chimericmolecule retains the specificity of the original immunoglobulin, despiteremoval of the constant regions and the introduction of a linkerpeptide. This modification usually leaves the specificity unaltered.These molecules were created historically to facilitate phage displaywhere it is highly convenient to express the antigen binding domain as asingle peptide. Alternatively, scFv can be created directly fromsubcloned heavy and light chains derived from a hybridoma. Single chainvariable fragments lack the constant Fc region found in completeantibody molecules, and thus, the common binding sites (e.g., proteinA/G) used to purify antibodies. These fragments can often bepurified/immobilized using Protein L since Protein L interacts with thevariable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promotingamino acid residues such as alaine, serine and glycine. However, otherresidues can function as well. Tang et al. (1996) used phage display asa means of rapidly selecting tailored linkers for single-chainantibodies (scFvs) from protein linker libraries. A random linkerlibrary was constructed in which the genes for the heavy and light chainvariable domains were linked by a segment encoding an 18-amino acidpolypeptide of variable composition. The scFv repertoire (approx. 5×10⁶different members) was displayed on filamentous phage and subjected toaffinity selection with hapten. The population of selected variantsexhibited significant increases in binding activity but retainedconsiderable sequence diversity. Screening 1054 individual variantssubsequently yielded a catalytically active scFv that was producedefficiently in soluble form. Sequence analysis revealed a conservedproline in the linker two residues after the V_(H) C terminus and anabundance of arginines and prolines at other positions as the onlycommon features of the selected tethers.

The recombinant antibodies of the present disclosure may also involvesequences or moieties that permit dimerization or multimerization of thereceptors. Such sequences include those derived from IgA, which permitformation of multimers in conjunction with the J-chain. Anothermultimerization domain is the Gal4 dimerization domain. In otherembodiments, the chains may be modified with agents such asbiotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created byjoining receptor light and heavy chains using a non-peptide linker orchemical unit. Generally, the light and heavy chains will be produced indistinct cells, purified, and subsequently linked together in anappropriate fashion (i.e., the N-terminus of the heavy chain beingattached to the C-terminus of the light chain via an appropriatechemical bridge).

Cross-linking reagents are used to form molecular bridges that tiefunctional groups of two different molecules, e.g., a stablizing andcoagulating agent. However, it is contemplated that dimers or multimersof the same analog or heteromeric complexes comprised of differentanalogs can be created. To link two different compounds in a step-wisemanner, hetero-bifunctional cross-linkers can be used that eliminateunwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo,preventing release of the targeting peptide prior to reaching the siteof action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Particular uses include adding a free amino or free sulfhydryl group toa protein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparate techniques.

E. Intrabodies

In a particular embodiment, the antibody is a recombinant antibody thatis suitable for action inside of a cell—such antibodies are known as“intrabodies.” These antibodies may interfere with target function by avariety of mechanism, such as by altering intracellular proteintrafficking, interfering with enzymatic function, and blockingprotein-protein or protein-DNA interactions. In many ways, theirstructures mimic or parallel those of single chain and single domainantibodies, discussed above. Indeed, single-transcript/single-chain isan important feature that permits intracellular expression in a targetcell, and also makes protein transit across cell membranes morefeasible. However, additional features are required.

The two major issues impacting the implementation of intrabodytherapeutic are delivery, including cell/tissue targeting, andstability. With respect to delivery, a variety of approaches have beenemployed, such as tissue-directed delivery, use of cell-type specificpromoters, viral-based delivery and use of cell-permeability/membranetranslocating peptides. With respect to the stability, the approach isgenerally to either screen by brute force, including methods thatinvolve phage diplay and may include sequence maturation or developmentof consensus sequences, or more directed modifications such as insertionstabilizing sequences (e.g., Fc regions, chaperone protein sequences,leucine zippers) and disulfide replacement/modification.

An additional feature that intrabodies may require is a signal forintracellular targeting. Vectors that can target intrabodies (or otherproteins) to subcellular regions such as the cytoplasm, nucleus,mitochondria and ER have been designed and are commercially available(Invitrogen Corp.; Persic et al., 1997).

By virtue of their ability to enter cells, intrabodies have additionaluses that other types of antibodies may not achieve. In the case of thepresent antibodies, the ability to interact with the MUC1 cytoplasmicdomain in a living cell may interfere with functions associated with theMUC1 CD, such as signaling functions (binding to other molecules) oroligomer formation. In particular, it is contemplated that suchantibodies can be used to inhibit MUC1 dimer formation.

F. Purification

In certain embodiments, the antibodies of the present disclosure may bepurified. The term “purified,” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein ispurified to any degree relative to its naturally-obtainable state. Apurified protein therefore also refers to a protein, free from theenvironment in which it may naturally occur. Where the term“substantially purified” is used, this designation will refer to acomposition in which the protein or peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the proteins in thecomposition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. Other methods for protein purification include,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; gel filtration, reversephase, hydroxylapatite and affinity chromatography; and combinations ofsuch and other techniques.

In purifying an antibody of the present disclosure, it may be desirableto express the polypeptide in a prokaryotic or eukaryotic expressionsystem and extract the protein using denaturing conditions. Thepolypeptide may be purified from other cellular components using anaffinity column, which binds to a tagged portion of the polypeptide. Asis generally known in the art, it is believed that the order ofconducting the various purification steps may be changed, or thatcertain steps may be omitted, and still result in a suitable method forthe preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e.,protein A) that bind the Fc portion of the antibody. Alternatively,antigens may be used to simultaneously purify and select appropriateantibodies. Such methods often utilize the selection agent bound to asupport, such as a column, filter or bead. The antibodies is bound to asupport, contaminants removed (e.g., washed away), and the antibodiesreleased by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. Another method forassessing the purity of a fraction is to calculate the specific activityof the fraction, to compare it to the specific activity of the initialextract, and to thus calculate the degree of purity. The actual unitsused to represent the amount of activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed protein or peptide exhibits adetectable activity.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

III. ACTIVE/PASSIVE IMMUNIZATION AND TREATMENT/PREVENTION OF EBOLA VIRUSINFECTION

The present disclosure provides pharmaceutical compositions comprisinganti-ebolavirus antibodies and antigens for generating the same. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of an antibody or a fragment thereof, or a peptide immunogen, anda pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a particular carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Other suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc. Examples of suitable pharmaceuticalagents are described in “Remington's Pharmaceutical Sciences.” Suchcompositions will contain a prophylactically or therapeuticallyeffective amount of the antibody or fragment thereof, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration, which can be oral,intravenous, intraarterial, intrabuccal, intranasal, nebulized,bronchial inhalation, or delivered by mechanical ventilation.

Active vaccines of the present disclosure, as described herein, can beformulated for parenteral administration, e.g., formulated for injectionvia the intradermal, intravenous, intramuscular, subcutaneous, or evenintraperitoneal routes. Administration by intradermal and intramuscularroutes are contemplated. The vaccine could alternatively be administeredby a topical route directly to the mucosa, for example by nasal drops,inhalation, or by nebulizer. Pharmaceutically acceptable salts, includethe acid salts and those which are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Passive transfer of antibodies, known as artificially acquired passiveimmunity, generally will involve the use of intravenous or intramuscularinjections. The forms of antibody can be human or animal blood plasma orserum, as pooled human immunoglobulin for intravenous (IVIG) orintramuscular (IG) use, as high-titer human IVIG or IG from immunized orfrom donors recovering from disease, and as monoclonal antibodies (MAb).Such immunity generally lasts for only a short period of time, and thereis also a potential risk for hypersensitivity reactions, and serumsickness, especially from gamma globulin of non-human origin. However,passive immunity provides immediate protection. The antibodies will beformulated in a carrier suitable for injection, i.e., sterile andsyringeable.

Generally, the ingredients of compositions of the disclosure aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water-free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

The compositions of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

IV. ANTIBODY CONJUGATES

Antibodies of the present disclosure may be linked to at least one agentto form an antibody conjugate. In order to increase the efficacy ofantibody molecules as diagnostic or therapeutic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety. Such a molecule or moiety may be, but is not limitedto, at least one effector or reporter molecule. Effector moleculescomprise molecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules which have been attached toantibodies include toxins, anti-tumor agents, therapeutic enzymes,radionuclides, antiviral agents, chelating agents, cytokines, growthfactors, and oligo- or polynucleotides. By contrast, a reporter moleculeis defined as any moiety which may be detected using an assay.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,photoaffinity molecules, colored particles or ligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging.” Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imagingmoieties used can be paramagnetic ions, radioactive isotopes,fluorochromes, NMR-detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present disclosure may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the disclosure may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentdisclosure are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and avidin andstreptavidin compounds. The use of such labels is well known to those ofskill in the art and are described, for example, in U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and4,366,241.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter and Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; Dholakia et al., 1989) and may be used as antibodybinding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may alsobe reacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors isachieved using monoclonal antibodies and the detectable imaging moietiesare bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

V. IMMUNODETECTION METHODS

In still further embodiments, the present disclosure concernsimmunodetection methods for binding, purifying, removing, quantifyingand otherwise generally detecting Ebolavirus and its associatedantigens. While such methods can be applied in a traditional sense,another use will be in quality control and monitoring of vaccine andother virus stocks, where antibodies according to the present disclosurecan be used to assess the amount or integrity (i.e., long termstability) of H1 antigens in viruses. Alternatively, the methods may beused to screen various antibodies for appropriate/desired reactivityprofiles.

Some immunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot to mention a few. In particular, a competitive assay forthe detection and quantitation of Ebolavirus antibodies directed tospecific parasite epitopes in samples also is provided. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle and Ben-Zeev (1999),Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al.(1987). In general, the immunobinding methods include obtaining a samplesuspected of containing Ebolavirus, and contacting the sample with afirst antibody in accordance with the present disclosure, as the casemay be, under conditions effective to allow the formation ofimmunocomplexes.

These methods include methods for purifying Ebolavirus or relatedantigens from a sample. The antibody will preferably be linked to asolid support, such as in the form of a column matrix, and the samplesuspected of containing the Ebolavirus or antigenic component will beapplied to the immobilized antibody. The unwanted components will bewashed from the column, leaving the Ebolavirus antigen immunocomplexedto the immobilized antibody, which is then collected by removing theorganism or antigen from the column.

The immunobinding methods also include methods for detecting andquantifying the amount of Ebolavirus or related components in a sampleand the detection and quantification of any immune complexes formedduring the binding process. Here, one would obtain a sample suspected ofcontaining Ebolavirus or its antigens, and contact the sample with anantibody that binds Ebolavirus or components thereof, followed bydetecting and quantifying the amount of immune complexes formed underthe specific conditions. In terms of antigen detection, the biologicalsample analyzed may be any sample that is suspected of containingEbolavirus or Ebolavirus antigen, such as a tissue section or specimen,a homogenized tissue extract, a biological fluid, including blood andserum, or a secretion, such as feces or urine.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to Ebolavirus orantigens present. After this time, the sample-antibody composition, suchas a tissue section, ELISA plate, dot blot or Western blot, willgenerally be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. Patents concerning the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149 and 4,366,241. Of course, one may find additionaladvantages through the use of a secondary binding ligand such as asecond antibody and/or a biotin/avidin ligand binding arrangement, as isknown in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding ligand, such as an antibody that hasbinding affinity for the antibody, is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firstbiotinylated antibody is used to detect the target antigen, and a secondantibody is then used to detect the biotin attached to the complexedbiotin. In that method, the sample to be tested is first incubated in asolution containing the first step antibody. If the target antigen ispresent, some of the antibody binds to the antigen to form abiotinylated antibody/antigen complex. The antibody/antigen complex isthen amplified by incubation in successive solutions of streptavidin (oravidin), biotinylated DNA, and/or complementary biotinylated DNA, witheach step adding additional biotin sites to the antibody/antigencomplex. The amplification steps are repeated until a suitable level ofamplification is achieved, at which point the sample is incubated in asolution containing the second step antibody against biotin. This secondstep antibody is labeled, as for example with an enzyme that can be usedto detect the presence of the antibody/antigen complex byhistoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the Ebolavirus or Ebolavirus antigen is added to the wells.After binding and washing to remove non-specifically bound immunecomplexes, the bound antigen may be detected. Detection may be achievedby the addition of another anti-Ebolavirus antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA.”Detection may also be achieved by the addition of a secondanti-Ebolavirus antibody, followed by the addition of a third antibodythat has binding affinity for the second antibody, with the thirdantibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theEbolavirus or Ebolavirus antigen are immobilized onto the well surfaceand then contacted with the anti-Ebolavirus antibodies of thedisclosure. After binding and washing to remove non-specifically boundimmune complexes, the bound anti-Ebolavirus antibodies are detected.Where the initial anti-Ebolavirus antibodies are linked to a detectablelabel, the immune complexes may be detected directly. Again, the immunecomplexes may be detected using a second antibody that has bindingaffinity for the first anti-Ebolavirus antibody, with the secondantibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

In another embodiment, the present disclosure contemplates the use ofcompetitive formats. This is particularly useful in the detection ofEbolavirus antibodies in sample. In competition based assays, an unknownamount of analyte or antibody is determined by its ability to displace aknown amount of labeled antibody or analyte. Thus, the quantifiable lossof a signal is an indication of the amount of unknown antibody oranalyte in a sample.

Here, the inventors propose the use of labeled Ebolavirus monoclonalantibodies to determine the amount of Ebolavirus antibodies in a sample.The basic format would include contacting a known amount of Ebolavirusmonoclonal antibody (linked to a detectable label) with Ebolavirusantigen or particle. The Ebolavirus antigen or organism is preferablyattached to a support. After binding of the labeled monoclonal antibodyto the support, the sample is added and incubated under conditionspermitting any unlabeled antibody in the sample to compete with, andhence displace, the labeled monoclonal antibody. By measuring either thelost label or the label remaining (and subtracting that from theoriginal amount of bound label), one can determine how much non-labeledantibody is bound to the support, and thus how much antibody was presentin the sample.

B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analyticaltechnique used to detect specific proteins in a given sample of tissuehomogenate or extract. It uses gel electrophoresis to separate native ordenatured proteins by the length of the polypeptide (denaturingconditions) or by the 3-D structure of the protein(native/non-denaturing conditions). The proteins are then transferred toa membrane (typically nitrocellulose or PVDF), where they are probed(detected) using antibodies specific to the target protein.

Samples may be taken from whole tissue or from cell culture. In mostcases, solid tissues are first broken down mechanically using a blender(for larger sample volumes), using a homogenizer (smaller volumes), orby sonication. Cells may also be broken open by one of the abovemechanical methods. However, it should be noted that bacteria, virus orenvironmental samples can be the source of protein and thus Westernblotting is not restricted to cellular studies only. Assorteddetergents, salts, and buffers may be employed to encourage lysis ofcells and to solubilize proteins. Protease and phosphatase inhibitorsare often added to prevent the digestion of the sample by its ownenzymes. Tissue preparation is often done at cold temperatures to avoidprotein denaturing.

The proteins of the sample are separated using gel electrophoresis.Separation of proteins may be by isoelectric point (pI), molecularweight, electric charge, or a combination of these factors. The natureof the separation depends on the treatment of the sample and the natureof the gel. This is a very useful way to determine a protein. It is alsopossible to use a two-dimensional (2-D) gel which spreads the proteinsfrom a single sample out in two dimensions. Proteins are separatedaccording to isoelectric point (pH at which they have neutral netcharge) in the first dimension, and according to their molecular weightin the second dimension.

In order to make the proteins accessible to antibody detection, they aremoved from within the gel onto a membrane made of nitrocellulose orpolyvinylidene difluoride (PVDF). The membrane is placed on top of thegel, and a stack of filter papers placed on top of that. The entirestack is placed in a buffer solution which moves up the paper bycapillary action, bringing the proteins with it. Another method fortransferring the proteins is called electroblotting and uses an electriccurrent to pull proteins from the gel into the PVDF or nitrocellulosemembrane. The proteins move from within the gel onto the membrane whilemaintaining the organization they had within the gel. As a result ofthis blotting process, the proteins are exposed on a thin surface layerfor detection (see below). Both varieties of membrane are chosen fortheir non-specific protein binding properties (i.e., binds all proteinsequally well). Protein binding is based upon hydrophobic interactions,as well as charged interactions between the membrane and protein.Nitrocellulose membranes are cheaper than PVDF, but are far more fragileand do not stand up well to repeated probings. The uniformity andoverall effectiveness of transfer of protein from the gel to themembrane can be checked by staining the membrane with CoomassieBrilliant Blue or Ponceau S dyes. Once transferred, proteins aredetected using labeled primary antibodies, or unlabeled primaryantibodies followed by indirect detection using labeled protein A orsecondary labeled antibodies binding to the Fc region of the primaryantibodies.

C. Immunohistochemistry

The antibodies of the present disclosure may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections fromthe capsule. Alternatively, whole frozen tissue samples may be used forserial section cuttings.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections. Again, whole tissue samples may besubstituted.

D. Immunodetection Kits

In still further embodiments, the present disclosure concernsimmunodetection kits for use with the immunodetection methods describedabove. As the antibodies may be used to detect Ebolavirus or Ebolavirusantigens, the antibodies may be included in the kit. The immunodetectionkits will thus comprise, in suitable container means, a first antibodythat binds to Ebolavirus or Ebolavirus antigen, and optionally animmunodetection reagent.

In certain embodiments, the Ebolavirus antibody may be pre-bound to asolid support, such as a column matrix and/or well of a microtitreplate. The immunodetection reagents of the kit may take any one of avariety of forms, including those detectable labels that are associatedwith or linked to the given antibody. Detectable labels that areassociated with or attached to a secondary binding ligand are alsocontemplated. Exemplary secondary ligands are those secondary antibodiesthat have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and all such labels may beemployed in connection with the present disclosure.

The kits may further comprise a suitably aliquoted composition of theEbolavirus or Ebolavirus antigens, whether labeled or unlabeled, as maybe used to prepare a standard curve for a detection assay. The kits maycontain antibody-label conjugates either in fully conjugated form, inthe form of intermediates, or as separate moieties to be conjugated bythe user of the kit. The components of the kits may be packaged eitherin aqueous media or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody may be placed, or preferably, suitably aliquoted. Thekits of the present disclosure will also typically include a means forcontaining the antibody, antigen, and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

VI. EXAMPLES

The following examples are included to demonstrate preferredembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof embodiments, and thus can be considered to constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of thedisclosure.

Example 1—Materials and Methods

Donors.

The donors were human subjects previously naturally infected with EbolaBundibugyo in Uganda in 2007 or U.S. citizens infected with Ebola 2014strain in 2014 in West Africa. Peripheral blood from the donors wasobtained months or years after the illness, when viral clearance hadbeen demonstrated, following informed consent. The study was approved bythe Vanderbilt University Institutional Review Board.

Viruses.

The recombinant Ebola Zaire strain Mayinga (EBOV) expressing eGFP wasgenerated in our laboratory by reverse genetics (Lubaki et al., 2013;Towner et al., 2005) from plasmids provided by the Special PathogensBranch at CDC and passaged 3 times in Vero E6 cells. For analysis ofantibody binding by ELISA, viruses were gamma-irradiated with the doseof 5×10⁶ rad. All work with Ebola virus was performed within theGalveston National Laboratory BSL-4 laboratories.

Generation of Human Hybridomas Secreting Monoclonal Antibodies (mAbs).

Peripheral blood mononuclear cells (PBMCs) from the donors were isolatedwith Ficoll-Histopaque by density gradient centrifugation. The cellswere cryopreserved immediately and stored in the vapor phase of liquidnitrogen until use. Previously cryopreserved samples were thawed, and 10million PBMCs were plated into 384-well plates (Nunc #164688) using: 17mL of cell culture medium (ClonaCell-HY Medium A, Stemcell Technologies#03801), 8 μg/mL of the TLR agonist CpG (phosphorothioate-modifiedoligodeoxynucleotide ZOEZOEZZZZZOEEZOEZZZT (SEQ ID NO: 81), Invitrogen),3 μg/mL Chk2 inhibitor (Sigma #C3742), 1 μg/mL cyclosporine A (Sigma#C1832) and 4.5 mL of clarified supernate from cultures of B95.8 cells(ATCC VR-1492) containing Epstein-Barr virus (EBV). After 7 days, cellsfrom each 384-well culture plate were expanded into four 96-well cultureplates (Falcon #353072) using cell culture medium containing 8 μg/mLCpG, 3 μg/mL Chk2i and 10 million irradiated heterologous human PBMCs(Nashville Red Cross) and incubated for an additional four days. Plateswere screened for Ebola virus antigen-specific antibody-secreting celllines using enzyme-linked immunosorbent assays (ELISAs). Cells fromwells with supernates reacting in an Ebola virus antigen ELISA werefused with HMMA2.5 myeloma cells using an established electrofusiontechnique (Yu et al., 2008). After fusion, hybridomas were resuspendedin medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μMthymidine (HAT Media Supplement, Sigma #H0262) and 7 μg/mL ouabain(Sigma #03125) and incubated for 18 days before screening hybridomas forantibody production by ELISA.

Human mAb and Fab Production and Purification.

After fusion with HMMA2.5 myeloma cells, hybridomas producingEbola-specific antibodies were cloned biologically by two rounds oflimiting dilution and by single-cell fluorescence-activated cellsorting. After cloning, hybridomas were expanded in post-fusion medium(ClonaCell-HY Medium E, STEMCELL Technologies #03805) until 50%confluent in 75-cm² flasks (Corning #430641). For antibody production,cells from one 75-cm² flask were collected with a cell scraper andexpanded to four 225-cm² flasks (Corning #431082) in serum-free medium(Hybridoma-SFM, Gibco #12045-076). After 21 days, supernates wereclarified by centrifugation and sterile filtered using 0.2-μm pore sizefilter devices. HiTrap Protein G or HiTrap MabSelectSure columns (GEHealthcare Life Sciences #17040501 and #11003494 respectively) were usedto purify antibodies from filtered supernates. Fab fragments weregenerated by papain digestion (Pierce Fab Preparation Kit, ThermoScientific #44985) and purified by chromatography using a two-columnsystem where the first column contained protein G resin (GE HealthcareLife Sciences #29048581) and the second column contained eitheranti-kappa or anti-lambda antibody light chain resins (GE HealthcareLife Sciences #17545811 and #17548211 respectively).

Screening ELISA.

ELISA plates were coated with recombinant Ebola virus proteins (20 μg in10 mL DPBS per plate) and incubated at 4° C. overnight. Plates wereblocked with 100 μL of blocking solution/well for 1 h. Blocking solutionconsisted of 10 g powdered milk, 10 mL of goat serum, 100 mL of 10×DPBS,and 0.5 mL of Tween-20 mixed to a 1 L final volume with distilled water.The presence of antibodies bound to the GP was determined using goatanti-human IgG horseradish peroxidase conjugated secondary antibodies(Southern Biotech #2040-05, 1:4,000 dilution) and 1-Step Ultra TMB-ELISAsubstrate (Thermo Scientific #34029), with optical density read at 450nM after stopping the reaction with 1M HCl.

Half Maximal Effective Concentration (EC₅₀) Binding Analysis.

Ebola virus GPs were coated onto 384-well plates (Thermo Scientific Nunc#265203) in DPBS at 2 μg/mL overnight, then antigen was removed andplates were blocked with blocking solution made as above. Antibodieswere applied to the plates using serial dilutions. The presence ofantibodies bound to the GP was determined using goat anti-human IgGalkaline phosphatase conjugate (Meridian Life Science #W99008A, 1:4,000dilution) and p-nitrophenol phosphate substrate tablets (Sigma #S0942),with optical density read at 405 nM after 120 minutes. A non-linearregression analysis was performed on the resulting curves using Prismversion 5 (GraphPad) to calculate EC₅₀ values.

Ebola Virus Neutralization Experiments.

Dilutions of mAbs in triplicate were mixed with Ebola virus or Ebolavirus expressing eGFP in MEM containing 10% FBS (HyClone), 50 μg/mLgentamicin (Cellgro #30-005-CR) with or without 50% guinea pigcomplement (MP Biomedicals #642836) in a total volume of 0.1 mL, andincubated for 1 hour at 37° C. for virus neutralization. Followingneutralization, virus-antibody mixtures were placed on monolayers ofVero E6 cells in 24-well plates, incubated for 1 hour at 37° C. forvirus adsorption, and overlayed with MEM containing 2% FBS and 0.8%methylcellulose (Sigma-Aldrich #M0512-1KG). After incubation for 5 days,medium was removed, cells were fixed with 10% formalin (FisherScientific #245-684), plates were sealed in plastic bags and incubatedfor 24 hours at room temperature. Sealed plates were taken out of theBSL-4 laboratory according to approved SOPs, and monolayers were washedthree times with phosphate buffered saline. Viral plaques wereimmunostained with a mAb against EBOV, clone 15H10 (BEI Resources#NR-12184). Alternatively, following virus adsorption, monolayers werecovered with MEM containing 10% FBS and 1.6% tragacanth (Sigma-Aldrich#G1128). After incubation for 14 days, medium was removed, cells werefixed with 10% formalin, plates were sealed in plastic bags, incubatedfor 24 hours at room temperature, and taken out of the BSL-4 laboratoryas above. Fixed monolayers were stained with 10% formalin containing0.25% crystal violet (Fisher Scientific #C581-100), and plaques werecounted. In some cases, when Ebola virus expressing eGFP, neutralizationwas scored using reduction of fluorescence.

Biolayer Interferometry Competition Binding Assay.

Biotinylated GP or GPΔmuc (EZ-Link® Micro NHS-PEG₄-Biotinylation Kit,Thermo Scientific #21955) (1 g/mL) was immobilized ontostreptavidin-coated biosensor tips (ForteBio #18-5019) for 2 minutes.After measuring the baseline signal in kinetics buffer (KB: 1×PBS, 0.01%BSA and 0.002% Tween 20) for two minutes, biosensor tips were immersedinto the wells containing primary antibody at a concentration of 100μg/mL for 10 minutes. Biosensors then were immersed into wellscontaining competing mAbs at a concentration of 100 μg/mL for 5 minutes.The percent binding of the competing mAb in the presence of the firstmAb was determined by comparing the maximal signal of competing mAbapplied after the first mAb complex to the maximal signal of competingmAb alone. MAbs were judged to compete for binding to the same site ifmaximum binding of the competing mAb was reduced to <30% of itsun-competed binding. MAbs were considered non-competing if maximumbinding of the competing mAb was >70% of its un-competed binding. Alevel of 30-70% of its un-competed binding was considered intermediatecompetition.

Sequence Analysis of Antibody Variable Region Genes.

Total cellular RNA was extracted from clonal hybridomas that producedEbola virus antibodies, and RT-PCR reaction was performed using mixturesof primers designed to amplify all heavy chain or light chain antibodyvariable regions. The generated PCR products were purified and clonedinto the pJet 1.2 plasmid vector (Thermo Scientific, #K1231) forsequence analysis. The nucleotide sequences of plasmid DNAs weredetermined using an ABI3700 automated DNA sequencer. Heavy chain orlight chain antibody variable region sequences were analyzed using theIMGT/V-Quest program (Brochet et al., 2008; Giudicelli et al., 2011).The analysis involved the identification of germline genes that wereused for antibody production, location of complementary determiningregions (CDRs) and framework regions (FRs) as well as the number andlocation of somatic mutations that occurred during affinity maturation.

Statistical Analysis.

EC₅₀ values for neutralization were determined by finding theconcentration of mAb at which a 50, reduction in plaque counts occurredafter incubation of virus with neutralizing antibody. A logistic curvewas fit to the data using the count as the outcome and thelog-concentration as the predictor variable. The results of the modelthen were transformed back to the concentration scale. Results arepresented as the concentration at the dilution that achieve a 50%reduction from challenge control with accompanying 95% confidenceintervals. Each antibody was treated as a distinct analysis in aBayesian non-linear regression model.

In Vivo Testing.

The animal protocol for testing of mAbs in mice was approved by theInstitutional Animal Care and Use Committee of the University of TexasMedical Branch at Galveston. BALB/c mice (Harlan) were placed in theABSL-4 facility of the Galveston National Laboratory. Groups of mice at5 animals per group were injected with individual mAbs by theintraperitoneal route. Untreated animals served as controls. For thechallenge, mice were injected with 1,000 PFU of the mouse-adapted Ebolavirus Mayinga strain by the intraperitoneal route. Animals were weighedand monitored daily over the three-week period after challenge. Onceanimals were symptomatic, they were examined twice per day. The diseasewas scored using the following parameters: dyspnea (possible scores0-5), recumbency (0-9), unresponsiveness (0-5), and bleeding/hemorrhage(0-5); the individual scores for each animal were summarized. Guinea pigstudies were conducted in a similar fashion.

Example 2—Results

Isolation of Monoclonal Antibodies (mAbs).

To generate human hybridoma cell lines secreting mAbs to Ebola virus GP,the inventors screened supernatants from EBV-transformed B cell linesderived from survivors of Ebola Bundibugyo virus (BDBV) in Uganda in2007 or of Ebolavirus ebola (EBOV) in the 2014 West African outbreak,for binding to several recombinant forms of Ebola GP or to irradiatedcell lysates prepared from Ebola virus-infected cell cultures. Theinventors fused transformed cells from B cell lines producing EbolaGP-reactive Abs to the Ebola GP antigens with myeloma cells andgenerated 90 cloned hybridomas secreting BDBV-reactive human mAbs fromBDBV survivors, and 119 cloned hybridomas secreting EBOV-reactive humanmAbs from EBOV survivors. The inventors screened for binding phenotypeagainst three types of glycoprotein, specifically, full-lengthglycoprotein, mucin deleted glycoprotein, and secreted glycoprotein.They found antibodies that bound with diverse patterns recognizing thevarious forms of glycoprotein, including clones that down to full-lengthand mucin delete a glycoprotein but did not have the potentialdeleterious property of binding to secreted like protein. The inventorsalso found antibodies that bound all three forms of glycoprotein.

Neutralization Activity.

To evaluate the inhibitory activity of the mAbs, the inventors firstperformed in vitro neutralization studies using a chimeric vesicularstomatitis virus with Ebola GP from on its surface (VSV/GP-Ebola) andlater they tested each of the antibodies for activity against live Ebolavirus strains. For the monoclonal antibodies isolated from BDBVsurvivors, 34 of the 90 BDBV mAb clones exhibited neutralizing activityin vitro. The inventors have found a similar proportion of neutralizingclones in the monoclonal antibodies isolated from survivors of the 2014Ebolavirus outbreak. Within clones isolated from the 2007 or 2014outbreak survivors, several of the antibodies isolated exhibit a higherpotency for neutralization then any monoclonal antibody of any speciesever reported for Ebola virus. The designated BDBV223 clone neutralizesBDBV with an IC₅₀ of 5 ng/mL remarkably, it also has the property ofcross-reactivity, and it neutralizes EBOV with an IC₅₀ of 50 ng/mL.Antibodies with this level of activity for neutralization have not beenreported previously. The inventors also found neutralizing clones fromsurvivors of the 2014 Ebola virus outbreak, including two clones thatneutralize with IC₅₀ of <1 μg/mL. The neutralization activity ofneutralizing Abs was in many cases enhanced by the presence ofcomplement.

Recognition of Varying Forms of GP.

To characterize the binding of isolated Abs to recombinant Ebola virusGPs, the inventors performed binding assays using either a recombinantMARV GP ectodomain containing the mucin-like domain (designated “fulllength GP” of just “GP”) or a recombinant GP lacking residues of themucin-like domain (GPΔmuc, from EBOV or BDBV or SUDV). Based on OD₄₀₅values at the highest Ab concentration tested (E_(max)) and 50%effective concentration (EC₅₀), the inventors divided the GP-specificAbs into different major phenotypic binding groups, based on bindingphenotype (designated Patterns 1, 2, 3 and 4). These distinctions areimportant, because they show some of the antibodies are morecross-reactive for diverse Ebola virus species than any antibodies everreported. They also showed that different antibodies recognize differentforms of glycoprotein, which may be useful. For example in some cases,one may find it desirable to have prophylactic or therapeutic antibodiesthat bind to full-length or mucin deleted glycoprotein, but avoidbinding to secreted glycoprotein. Alternatively, if one is seeking toprevent pathogenic effects mediated by the secreted glycoprotein, onewould seek antibodies that retained binding to the secreted form ofglycoprotein. The inventors have isolated antibodies with both types ofbinding patterns, and with differing levels of cross-reactivity fordiverse Ebola virus species.

Competition-Binding Studies.

To determine whether mAbs from distinct binding groups targeteddifferent antigenic regions on the Ebola virus GP surface, the inventorsperformed competition-binding assays using a real-time biosensor. Theytested diverse nAbs from our panel of Ebola virus antibodies in a tandemblocking assay in which biotinylated GP was attached to a streptavidinbiosensor. The inventors identified several major competition bindinggroups within their antibodies, and subsequent electron microscopystudies of antigen antibody complexes show that one group binds to theglycan cap region on the glycoprotein, and another 2 groups bind loweron the glycoprotein, one at the base and one group lower down at theheptad repeat 2 region. These data suggested that these neutralizing Abstarget at least three major antigenic regions on the Ebola virus GPsurface.

Electron Microscopy Studies of Antigen-Antibody Complexes.

To determine the location of the antigenic region targeted by Ebolavirus neutralizing Abs, the inventors performed with collaboratorsnegative stain single-particle electron microscopy (EM) studies usingcomplexes of GP with Fab fragments of neutralizing Abs. The EMreconstructions showed that Fab fragments for one competition bindingpattern group of neutralizing Abs bind at the top of the GP in or nearthe glycan cap site. A second competition binding pattern group ofneutralizing Abs bind at the bottom of the GP in or near what would be aputative MPER or heptad repeat 2 region.

Cross-Reactive Binding of Ebola Virus Antibodies with Diverse Species ofGP.

It is surprising that human MARV neutralizing Abs recognize GP fromdiverse species of Ebola, since previously reported murine mAbs and onehuman phage display library derived antibody (KZ52) exhibited a bindingpattern that was restricted to a single species of Ebola virus, Todetermine whether the isolated Ebola virus neutralizing Abs could bindin a cross-reactive manner to diverse Ebola virus species, the inventorsperformed ELISA binding assays using recombinant forms of BDBV, EBOV,and SUDV GPs. Several of the Ebola virus antibodies neutralizing Absrecognized two or even three species Ebola GP. They tested the breadthof neutralization of MARV neutralizing Abs for filoviruses using a panelof different Ebola virus isolates. Some of the neutralizing Absneutralized diverse Ebola virus species, which is a newly discovered anddesirable property.

In Vivo Testing.

The inventors tested the in vivo protective activity of the mAbs inmurine and guinea pig models of infection using mouse- or guinea-pigadapted Ebola Zaire Mayinga strain. Inoculation of mice or guinea pigswith live Ebola virus caused clinical disease, and in a proportion orall of animals caused lethal disease. They selected five of the BDBVsurvivor mAbs among those with in vitro neutralization IC₅₀ values anddiverse properties: BDBV223, BDBV270, and BDBV289, BDBV317, BDBV324.When used as monotherapy and given at 24 hours after lethal inoculation,each exhibited a marked therapeutic effect. The inventors also testedcombinations BDBV223+BDBV270, or BDBV223+BDBV289, since BDBV223recognizes the MPER region and the other two (BDBV270 and BDBV289)recognize the glycan. Each of the combinations exhibited increasedtherapeutic effect. They also tested the most potent antibody (BDBV223)as monotherapy but given as a two-dose treatment (at 1 day and 3 dayafter lethal inoculation). This two-dose regimen appeared more effectivethan single dose therapy.

Example 3—Discussion

There is an obvious urgent need for prophylactic and therapeuticinterventions for filovirus infections given the massive outbreak ofEBOV infections in West Africa in 2014. There is very little informationabout the structural determinants of neutralization on which to base therational selection of antibodies, and for Ebola virus there have been noreported human neutralizing Abs with naturally paired heavy and lightantibody chains.

For the three most important species, EBOV and BDBV and SUDV, theinventors studied survivors of the first two species, EBOV and BDBV andobtained human monoclonal antibodies from survivors of each of those twoinfections. Ninety human monoclonal antibodies that obtained from eightsurvivors of BDBV infection in Uganda in 2007. They have also obtainedcells from U.S. survivors who were infected while working in West Africain 2014. From one of those individuals, they obtained about 93 new humanmonoclonal antibodies that were induced by the current strain, EBOV,historically called the Zaire strain. Additional donors have yielded 26more clones from EBOV immune donors. Data show that some of theseantibodies have neutralizing activity against the EBOV (20 of the ˜50mAbs tested from the one individual so far for neutralizing activity).Thus, in summary, the inventors have two sets of antibodies, one set of90 mAbs induced by BDBV infection and one set of 119 mAbs induced byEBOV infection.

In conclusion, this study reveals that naturally-occurring human Ebolaneutralizing Abs isolated from the B cells of recovered donors targetseveral antigenic sites on Ebola virus GP, suggesting that at least twomajor mechanisms of Ebola virus neutralization. Remarkably, some of theisolated antibodies bound not only to the inducing virus (BDBV or EBOV)but also exhibited cross-reactive binding to other GPs, including BDBV,EBOV and SUDV GP. This information can be used to inform development ofnew therapeutics and structure-based vaccine designs againstfiloviruses. Furthermore, as these neutralizing mAbs are fully human andexhibit inhibitory activity, they could be formulated as components of aprophylactic or therapeutic approach for filovirus infection anddisease. Indeed, challenge studies using murine and guinea pig modeldhere show clear evidence of in vivo activity. Their ability to bind abroad range of Ebola virus isolates indicates they may offer detectionof or efficacy against new viral strains yet to emerge. Since these mAbsbind to diverse forms of Ebola virus GP, these antibodies could beselected for preferred activity in vivo, for instance avoiding bindingto secreted GP or including binding to secreted GP.

TABLE 1A Biological properties of human mAbs isolated from a 2014survivor of West African outbreak: in vivo protection, binding to GP ofvarious filovirus species, and epitope mapping EBOV49 EBOV52 EBOV62EBOV63 EBOV82 EBOV87 EBOV90 EBOV95 Mice 80% 0% 60% 0% 80% 20% 100% 0%protection EBOV 262 712 228 283 36 724 67 129 GP binding (EC50, μg/ml)BDBV — 1838 508 — — 1389 — — GP binding (EC50, μg/ml) SUDV — — — — — 629— — GP binding (EC50, μg/ml) EBOV 61 82 51 38 21 51 25 41 sGP binding(EC50, μg/ml) Location 252Phe→Cys, 275Trp→Gly, 234Phe→VaL 155Lys→Arg,224Gly→Asp, 267Ser→Asn, 227Thr→Ile, 280Glu→Lys, of escape glycan capglycan cap 273Leu→Pro, receptor- glycan cap 271Gly→Glu, glycan capglycan cap mutations 308Phe→Leu, binding glycan cap glycan cap; domain;365Leu→Pro, 280Glu→Lys, 368Leu→Pro, glycan cap 394Tyr→His, mucin-likedomain

TABLE 2 CDR sequences Antibody CDRH1 CDRH2 CDRH3 BDBV43 DSFSRKYGIMPIVGLT ARDEIIGARPHWFDS (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35)BDBV223 GGSFTTTY VNYSGNA TSRIRSHIAYSWKGDV (SEQ ID NO: 36)(SEQ ID NO: 37) (SEQ ID NO: 38) EBOV9 GGTFSSYT IIPKLGIALYYCARVLLSSRDAFDIW (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 41)EBOV49 GFTFSSYE ISSSGRTI AREPYVDGILYGAGDSYFDY (SEQ ID NO: 42)(SEQ ID NO: 43) (SEQ ID NO: 44) EBOV52 GGSISSYY IYDSGRTASLGPFDKLWFGELLPGWFDP (SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47)EBOV63 GFTLNFYN ISSSSNYI ARDFVQLLIPQRDEWQGVHDYYGMDV (SEQ ID NO: 48)(SEQ ID NO: 49) (SEQ ID NO: 50) EBOV82 GFTFTNAW IKSNTDGGTTTTGKSDCSGGNCYVVDY (SEQ ID NO: 51) (SEQ ID NO: 52) (SEQ ID NO: 53) EBOV90GFTFSNAW IKSKNDGGTA ITFLRPDH (SEQ ID NO: 54) (SEQ ID NO: 55)(SEQ ID NO: 56) EBOV109 GYTFTGYY INPNSGGT CATNKGTNGRYYYYGMDVW(SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) EBOV62 GFTFSSYW IKQDGSAKARDGLLGISDLLYPIYYFDY (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84)EBOV87 GYTFTSYA ISGNNGNT ARDADIVVVVGATGTYYYGMDV (SEQ ID NO: 85)(SEQ ID NO: 86) (SEQ ID NO: 87) EBOV157 GFTVSNNY FYSDGTT ARQASGYDAYYMDV(SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO: 90) BDBV289 GATFGSDTIIPFFGEA ARQINEMATFGEIHYYTYMDV (SEQ ID NO: 91) (SEQ ID NO: 92)(SEQ ID NO: 93) BDBV231 SDSIRSYS IYYSGNI ARDWITIFGRYFDV (SEQ ID NO: 94)(SEQ ID NO: 95) (SEQ ID NO: 96) BDBV275 GFNFGDYV IRGKTFGATT TRRATSTWYEDY(SEQ ID NO: 97) (SEQ ID NO: 98) (SEQ ID NO: 99) BDBV315 GDSISSGSYYIYTSGST ARDPITIFGGVIFGWGMDV (SEQ ID NO: 100) (SEQ ID NO: 101)(SEQ ID NO: 102) BDBV329 GGTFDTYA IIPVLGIVARGLRSLSPRGQEGPTPAPGWRRAQYHYYYMDV (SEQ ID NO: 103) (SEQ ID NO: 104)(SEQ ID NO: 105) BDBV335 GGSINSDSYY VYTSGST ARVVWGSYRSYHYSYGMDV(SEQ ID NO: 106) (SEQ ID NO: 108) (SEQ ID NO: 109) BDBV354 GYAFTTYAISTYYGTT VRDRSWLATSRPYDAFDI (SEQ ID NO: 110) (SEQ ID NO: 111)(SEQ ID NO: 112) BDBV386 GGSISSGRFY IYTSGST ATELYYYGSGSYDPLWS(SEQ ID NO: 113) (SEQ ID NO: 114) (SEQ ID NO: 115) BDBV397 GGSISSGSYFIYTSGTT ATSPYYYDSSHYYDY (SEQ ID NO: 116) (SEQ ID NO: 117)(SEQ ID NO: 118) BDBV399 GGSISNGGYH IYYSGST ARDRIRGGPIDY(SEQ ID NO: 119) (SEQ ID NO: 120) (SEQ ID NO: 121) BDBV353 GYTFSDYYINPYSGGT ARLYGAGSHYNHYNGMDV (SEQ ID NO: 122) (SEQ ID NO: 123)(SEQ ID NO: 124) BDBV410 GGSVSSGRYF IHSSGRT <Not yet available>(SEQ ID NO: 125) (SEQ ID NO: 126) BDBV270 GASISRGLYY IYTSGSIVRDAPWGDFLTGYFGFYGMDV (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 129)BDBV324 GYTFTSFE MNPKSGDT ARGPHVGEVVPGLMAGTYYFPLDV (SEQ ID NO: 130)(SEQ ID NO: 131) (SEQ ID NO: 132) BDBV403 GGTFSNSI IIPIVGLV AINGVNIPDTLT(SEQ ID NO: 133) (SEQ ID NO: 134) (SEQ ID NO: 135) BDBV407 GGSIRSYFIYYSGRP ARDERLLVEVGTDHFYYGLDV (SEQ ID NO: 136) (SEQ ID NO: 137)(SEQ ID NO: 138) BDBV425 GYTFTSFG INTYNGDT ARDSHLISIAVANTPNDF(SEQ ID NO: 139) (SEQ ID NO: 140) (SEQ ID NO: 141) BDBV426 GGSISSDDRYIYYSGST ATVTAYSPATMIVVGTEHGFDY (SEQ ID NO: 142) (SEQ ID NO: 143)(SEQ ID NO: 144) BDBV317 GLTFSNFG IRFDGSNK GRVLYGAAADF (SEQ ID NO: 145)(SEQ ID NO: 146) (SEQ ID NO: 147) BDBV342 GGTFSSYA IIPIFGKPARGQGEIVVMVGHDDGGDYLGY (SEQ ID NO: 148) (SEQ ID NO: 149)(SEQ ID NO: 150) BDBV357 GGSISGSI ISLSGST ARHRKSSKMVRGIEVFYYYYMDV(SEQ ID NO: 151) (SEQ ID NO: 152) (SEQ ID NO: 153) BDBV340 GGSISSGSFYFYTTGST ARGPVSYYSGNLYYFDY (SEQ ID NO: 154) (SEQID NO: 155)(SEQ ID NO: 156) BDBV392 GFTFSSFG IRYDGSDK AKRGGHDYGYYDNNRYIDL(SEQ ID NO: 157) (SEQ ID NO: 158) (SEQ ID NO: 159) BDBV415 GGTFSSYGIIPKFATA AGHFPQRKPITTIVVITYWSLDL (SEQ ID NO: 160) (SEQ ID NO: 161)(SEQ ID NO: 162) BDBV343 GVTFSRYT ISPILGTA ARDAPIILVEGPETGMDV(SEQ ID NO: 163) (SEQ ID NO: 164) (SEQ ID NO: 165) BDBV377 GFTFNSYGIWFDGSKK AKDLLYGSGMVPNYYYYGLDV (SEQ ID NO: 166) (SEQ ID NO: 167)(SEQ ID NO: 168) CDRL1 CDRL2 CDRL3 BDBV43 QSVSSN GSS LQYYNWPRT(SEQ ID NO: 60) (SEQ ID NO: 61) BDBV223 QSVPRNY GAS HQYDRLPYT(SEQ ID NO: 62) (SEQ ID NO: 63) EBOV49 QSISNY AAS QQSYNTPPVT(SEQ ID NO: 64) (SEQ ID NO: 65) EBOV52 QSVHNY DAS QHRSNWLT(SEQ ID NO: 66) (SEQ ID NO: 67) EBOV63 QSVSNSY GAS QHYGSSQLT(SEQ ID NO: 68) (SEQ ID NO: 69) EBOV109 QSLLHSNGYNY LGS CMQALQTITF(SEQ ID NO: 70) (SEQ ID NO: 71) EBOV62 QNIGSY AAS QQSYSIPRT(SEQ ID NO: 169) (SEQ ID NO: 170) EBOV87 QSISSW DAS QQYKSSLRT(SEQ ID NO: 171) (SEQ ID NO: 172) EBOV157 QSINSW QAS QQYSSFPLT(SEQ ID NO: 173) (SEQ ID NO: 174) BDBV41 QSITSTY GAS QQYHSSL(SEQ ID NO: 175) (SEQ ID NO: 176) BDBV231 QNLLYSSNNKNF WAS QQYYTIPPT(SEQ ID NO: 177) (SEQ ID NO: 178) BDBV275 QSVLYTPNNHNY WAS QQYHIPPYS(SEQ ID NO: 179) (SEQ ID NO: 180) BDBV315 QTLLHSNGYNY LGS MQALQTPVT(SEQ ID NO: 181) (SEQ ID NO: 182) BDBV329 QSVSSNY GAS QQYGSSPGT(SEQ ID NO: 183) (SEQ ID NO: 184) BDBV335 QSVGSSY GAS QQSGSSPET(SEQ ID NO: 185) (SEQ ID NO: 186) BDBV354 QDISST GAS QHFYYFPRT(SEQ ID NO: 187) (SEQ ID NO: 188) BDBV386 QGINNN DAS QQNANLPHT(SEQ ID NO: 189) (SEQ ID NO: 190) BDBV397 QDITNY DAS QQSADLPLT(SEQ ID NO: 191) (SEQ ID NO: 192) BDBV399 QGIDNY AAS QRYNLAPSA(SEQ ID NO: 193) (SEQ ID NO: 194) BDBV353 QSIGSL RAS QQFNSY(SEQ ID NO: 195) (SEQ ID NO: 196) BDBV410 QSLLHSNGETY EVS MQSVLLPYT(SEQ ID NO: 197) (SEQ ID NO: 198) BDBV270 QSINTY AAS QQSFTTPYT(SEQ ID NO: 199) (SEQ ID NO: 200) BDBV324 QSISRW KVS QQYDTYPWT(SEQ ID NO: 201) (SEQ ID NO: 202) BDBV403 QSVSSSY DAS QQYGSSAIT(SEQ ID NO: 203) (SEQ ID NO: 204) BDBV407 QSLLHSNGYNF LGS MQA(SEQ ID NO: 205) BDBV425 QSVYSYY DAS QYYGNSHQGAA (SEQ ID NO: 206)(SEQ ID NO: 207) BDBV317 HSLLYSSDNKNY WAS QQYYTKSFT (SEQ ID NO: 208)(SEQ ID NO: 209) BDBV342 SSNVGAYNY DVT YSYAGSYTWI (SEQ ID NO: 210)(SEQ ID NO: 211) BDBV357 ISDIGGYDY DVS SSYTRTYTPHVV (SEQ ID NO: 212)(SEQ ID NO: 213) BDBV340 SSDIGNNNY DVN SSYTNNRTFS (SEQ ID NO: 214)(SEQ ID NO: 215) BDBV259 KLGDRY QDT QAWDTS (SEQ ID NO: 216)(SEQ ID NO: 217) BDBV343 NSDVGGYDY DVN CSYAGSYTFV (SEQ ID NO: 218)(SEQ ID NO: 219)

TABLE 3 Variable Region Protein Sequences BDBV43 VH (SEQ ID NO: 1)QVQLVQSGAEVKKPGSSVKVSCRASGDSFSRKYGISWVRQAPGQGFEWMGTIMPIVGLTTSAQKFQGRVTITADKSTSTAHMELNSLTSEDTAIYYCARDEIIGARPHWFDSWGQGTLVTVSS BDBV43 VL (SEQ ID NO: 2)EIVMTQSPAIMSVSPGKRATLSCRASQSVSSNLAWYQRKPGQAPRLLIYGSSTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQYYNWPRTFGQ GTKVEIK BDBV223 VH(SEQ ID NO: 3) QVQLQQWGAGLLKPSETLSLTCAVYGGSFTTTYWNWIRQPPGKGLEWIGEVNYSGNANYNPSLKGRVAISVDTSKNQFSLRLNSVTAADTAIYYCTSRIRSHIAYSWKGDVWGKGTTVTVSS BDBV223 VL (SEQ ID NO: 4)EIVMTQSPGTLSLSPGERATLSCRASQSVPRNYIGWFQQKPGQAPRLLIYGASSRAAGFPDRFSGSGSGTDFTLTITRLEPEDFAMYYCHQYDRLPYTFG QGTKLEIK  EBOV49 VH(SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEINWVRQAPGRGLEWVSYISSSGRTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREPYVDGILYGAGDSYFDYWGQGTLVTVSS EBOV49 VL (SEQ ID NO: 6)DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKVLIYAASSLQSGVSSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSYNTPPVTFG QGTRLEIK EBOV52 VH(SEQ ID NO: 7) QVQLQESGPGLVKPSETLSLNCTVSGGSISSYYWSWIRQPPQKGLEWIGYIYDSGRTKYNPSLKSRVTISLDTSKNQFSLKLSSVTAADTAVYYCASLGPFDKLWFGELLPGWFDPWGQGTLVTVSS EBOV52 VL (SEQ ID NO: 8)EIVLTQSPATLSLSPGGRATLSCRASQSVHNYLAWYQQKSGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPDDFAVYYCQHRSIYWLTFGG GTKVEIK EBOV63 VH(SEQ ID NO: 9) EVQLVESGGGLVKPGGSLRLSCAASGFTLNFYNMNWVRQAPGKGLEWVSSISSSSNYIYYADSVKGRFTISRDNARKSLYLQMNSLRAEDTAVYYCARDFVQLLIPQRDEWQGVHDYYGMDVWGQGTLVTVSS EBOV63 VL (SEQ ID NO: 10)EIVLTQSPGTLSLSPGGRATLSCRASQSVSNSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQHYGSSQLTFG GGTKVEIK EBOV82 VH(SEQ ID NO: 11) EVQLVESGGGLVKPGGSLRLSCAASGFTFTNAWMNWVRQAPGKGLEWVGRIKSNTDCGTTDYAAPVKGRFTISRDDSKKTLYLQMNSLKTEDTAVYYCTTGKSDCSGGNCYVVDYWGQGTLVTVSS EBOV82 VL <Not Yet Available> EBOV90 VH(SEQ ID NO: 12) EVQLVESGGGLVKPGGALRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKNDGGTADYAAPVKGRFSISRDDSKNILYLQMNSLKIEDTAVYYCIT FLRPDHWGQGTLVTVSSEBOV90 VL <Not Yet Available> EBOV9 VH (SEQ ID NO: 72)QVQLVQSGVEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPKLGIANYAQNAQKFQGRVTITADKSTSTAYMELSRLRSEDTALYYCARVLLSSRDAFDIWGQGTLVTVSS EBOV9 VL <Not Yet Available> EBOV109 VH(SEQ ID NO: 73) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGWVTMTRDTSTSTAYMELRRLRSDDTAVYYCATNKGTNGRYYYYGMDVWGQGTLVTVSS EBOV109 VL (SEQ ID NO: 74)DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTI TFGQGTRLEIKEBOV119 VH (SEQ ID NO: 75)QVQLVQSGVEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPKLGIANYAQNAQKFQGRVTITADKSTSTAYMELSRLRSEDTALYYCA RVLLSSRDAFDIWGQGTLVTEBOV119 VL <Not Yet Available> EBOV62_VH (SEQ ID NO: 220)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSAKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGLLGISDLLYPIYYFDYWGQGTLVTVSS EBOV62_VL (SEQ ID NO: 221)DIVMTQSPSSLSASVGDRVTITCRASQN1GSYLNWYQQKPGKAPNLLMYAASSLQSGVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSIPRTFGQ GTQLEIK >EBOV87_VH(SEQ ID NO: 222) QVQLVQSGAEVKRPGASVKVSCKASGYTFTSYAISWVRQAPGQGLEWMGWISGNNGNTNYAQKLQGRLTMTTDTSTSTAYMELRSLRSDDTAVYYCARDADIVVVVGATGTYYYGMDVWGQGTLVTVSS >EBOV87_VL (SEQ ID NO: 223)DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYKSSLRTFGQ GTQLEIK EBOV157_VH(SEQ ID NO: 224) QVQLVQSGGGLVQPGGSLRLSCAASGFTVSNNYMSWVRQAPGKGLEWVSIFYSDGTTYNADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQAS GYDAYYMDVWGQGTLVTVSSEBOV157_VL (SEQ ID NO: 225)DIVMTQSPSTLSASVGDRVTITCRASQSINSWLAWYQQRPGKAPKLLIYQASTLERGVPSRFSGSGAGTEFTLTISSLQPDDFATYYCQQYSSFPLTFGG GTKVELK BDBV289_VH(SEQ ID NO: 226) QVQLVQSGAEVKKPGSSVKVSCKASGATFGSDTVTWVRQAPGQGLEWMGGIIPFFGEANYAQRFQGRVTITADKSTNTAYMELSSLRSEDTAVYFCARQINEMATFGEIHYYTYMDVWGQGTLVTVSS BDBV289_VL (SEQ ID NO: 227)GSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPRQAPVLVFYGKNNRPSGIPDRFSGSSSGNTASLTISGAQAEDEADYYCNSRDSSSNHLVFG GGTKLTVLS BDBV41<Not Yet Available> BDBV41_VL (SEQ ID NO: 228)EIVMTQSPGTLSLSPGERATLSCRASQSITSTYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHSSLFGGG TKVEIK BDBV231_VH(SEQ ID NO: 229) QVQLVQSGPGLVKPSETLSLTCTVSSDSIRSYSWSWLRQPPGKGLEWIGFIYYSGNINYNPSLKSRVTISVDTSKNQLSLNLSSVTAADTAVYYCARDWI TIFGRYFDVWGRGTLVTVSSBDBV231_VL (SEQ ID NO: 230)DIVMTQSPDSLAVSLGERATINCKSSQNLLYSSNNKNFLTWYQHKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVALYYCQQYYTI PPTFGQGTKVEIKBDBV275_VH (SEQ ID NO: 231)QVQLVQSGGGFVQPGRSLRLSCTASGFNFGDYVMSWVRQAPGKGLEWVGFIRGKTFGATTEYAASVKGRFTISRDDSKSIAYLQIKSLKTEDTAVYYCTR RATSTWYEDYWGQGTLVTVSSBDBV275_VL (SEQ ID NO: 232)DIVMTQSPDSLAVSLGERATTNCKSSQSVLYTPNNHNYLAWYQQKPGQPPKLLIYWASAREPGVPDRFSGSCSGTDFTLTISSLQAEDVAVYYCQQYHIP PYSFGQGTKLEIKBDBV315_VH (SEQ ID NO: 233)QVQLVQSGPGLVKPSQTLSLTCTVSGDSISSGSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTISVDTSKNQFSLNLSSVTAADTAVYYCARDPITIFGGVIFGWGMDVWGQGTLVTVSS BDBV315_VL (SEQ ID NO: 234)DIVMTQSPLSLPVTPGEPASISCRSSQTLLIISNGYNYLYWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTEKISRVEAEDVGVYYCMQALQT PVTFGPGTKVDIKBDBV329_VH (SEQ ID NO: 235)QVQLVQSCAEVKKPGSSVKVSCKASGGTFDTYAISWVRQAPGQGLEWMGGIIPVLGIVDYAQKFQGRVTITAAKFTNIAYMELSSLRSEDAAVYYCARGLRSLSPRGQEGPTPAPGWRRAQYHYYYMDVWGTGTLVTVSS BDBV329_VL (SEQ ID NO: 236)EIVMTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGPDFTLTISRLEPEDFAVYYCQQYGSSPGTFG GGTKVEIK  BDBV335_VH(SEQ ID NO: 237) QVQLQESGPGLVRPSQTLSLTCTVSGGSINSDSYYWNWIRQPAGKGLEWLGRVYTSGSTNYNPSLKSRVTISVDTSKNQVSLRLNSVTAADTGVYYCARVVWGSYRSYHYSYGMDVWGQGTLVTVSS BDBV335_VL (SEQ ID NO: 238)EIVMTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQRPGQAPRLLFYGASYRATGIPDRFSASGSGTDFSLTINRLEPEDFAVYYCQQSGSSPETFG QGTKLEIK BDBV354_VH(SEQ ID NO: 239) QVQLVQSGVEVKKPGASVKVSCKASGYAFTTYAISWVRQAPGQGLEWMGWISTYYGTTYYAQNLQGRVTMTTDTSTSTSYLELRSLRSDDTAWYCVRDRSWLATSRPYDAFDIWGQGTLVTVSS BDBV354_VL (SEQ ID NO: 240)AIQMTQSPSSLSASVGDRVTITCRASQDISSTLAWYQQKPGKAPKLLIYGASSLESGVPSRFNGSGSGTDFTLTISSLQPEDFATYYCQHFYYFPRTFGQ GTRLEIR BDBV386_VH(SEQ ID NO: 241) QVQLVQSGPGLVKPSQTLSLTCTVSGGSISSGRFYWSWVRQPAGRGLEWIGRIYTSGSTNYNPSLKSRVSISVDTSKNQFSLKLSSVTAADTAVYYCATELYYYGSGSYDPLWSWGQGTLVTVSS BDBV386_VL (SEQ ID NO: 242)DIVMTQSPSSLSASVGDRVTITCQASQGINNNLNWHQQKPGKAPKLLIYDASNLERGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQNANLPFITFG QGTKLEIK BDBV397_VH(SEQ ID NO: 243) QVQLVQSGPGLVKPSQTLSLTCTVSGGSISSGSYFWNWIRQPAGKGLEWIGRIYTSGTTNYNPSLRSRLTISVDTSKNQFSLKLNSVTAADTAVYYCATSPYYYDSSHYYDYWGQGTLVTVSS BDBV397_VL (SEQ ID NO: 244)DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIFDASNLEKGVPSRFSATGSATDFTFTISSLQPEDTATYYCQQSADLPLTFGQ GTRLDIK BDBV399_VH(SEQ ID NO: 245) QVQLVQSGPGLVKPSQTLSLTCNVSGGSISNGGYHWSWIRQVPGKGLEWIGHIYYSGSTSYTPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARD RIRGGPIDYWGQGTLVTVSSBDBV399_VL (SEQ ID NO: 246)DIQMTQSPSSLSASVGERVTITCRASQGIDNYLAWYQQKPGKVPKLLIYAASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNLAPSAFGQ GTKVEIR BDBV353_VH(SEQ ID NO: 247) QVQLVQSGAEMRKPGASVKVSCKASGYTFSDYYIHWVRQAPGQGLEWEGWINPYSGGTNYAQKFQGRVTMIRDTSISTAHMELSGLRSDDTALYFCARLYGAGSHYNHYNGMDVWGQGTLVTVSS BDBV353_VL (SEQ ID NO: 248)DIQMIQSPSILSASVGDRVIITCRASQSIGSLLAWYQQKPGKAPKLLIYRASTLQGGVPSRFSGSGSGTEFTLTISSLQPDDVATYYCQQFNSYFGGGTK VEIK BDBV410_VH(SEQ ID NO: 249) QVQLQQSGPGLVRPSQTLSLTCSVSGGSVSSGRVFWNWIRQSAGKGLEWIGRIHSSGRTNSNPSLKSRVTISVDTSKNQFSLHLGSVTAADTAVYYCAR BDBV410_VL(SEQ ID NO: 250) DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSNGETYLFWYEQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSVLLP YTFGQGTKLEIKBDBV270_VH (SEQ ID NO: 251)QVQLQESGPGLVKPSQTLSLTCTVSGASISRGLYYWSWIRQPAGKGLEWIGRIYTSGSLNYNPSLKSRVTISVDTSKNQFSLRLSSVIATDTAVYYCVRDAPWGDFLTGYFGFYGMDVWGQGTLVTVSS BDBV270_VL (SEQ ID NO: 252)DIVMTQSPSSLSASVGDRVTITCRASQSINTYLNWYQQKPGKAPKFLIYAASSLHSGVPSRFSGSGSGTDFTLTINSLQPDDFATYYCQQSFTTPYTFGQ GTKLEIK BDBV324_VH(SEQ ID NO: 253) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSFEIHWVRQGSGQGLEWMGRMNPKSGDTVSAQKFQGRVTLTRDTSINAAYMELGSLSSEDTAVYYCARGPHVGEVVPGLMAGTYYFPLDVWGQGTLVTVSS BDBV324_VL (SEQ ID NO: 254)DIQMTQSPSTLSASIGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYKVSDLQSGVPSRFSGSGYGTEFTLTIGSLQPDDLATYYCQQYDTYPWTFGQ GTKLEIK BDBV403_VH(SEQ ID NO: 255) QVQLVQSGAEVKKPGSSVKVSCNASGGTFSNSILNWVRQAPGQGLEWMGRIIPIVGLVNFAQKFEGRVTFTADKFTNTAYMELNSLRFEDTAVYYCAING GKYPGYFDYWGQGTLVTVSSBDBV403_VL (SEQ ID NO: 256)DIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQHQPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLIISRLEPEDFAVYYCQQYGSSAITFG QGTRLEIK BDB407_VH(SEQ ID NO: 257) QVQLQESGPGLVKPSETLSLTCAVSGGSIRSYFWSWIRQAPGKGLEWIGNIYYSGRPNYNPSLKNRVTISADTSNNEVSLELSAVTAADTAVYFCARDERLLVEVGTDHFYYGLDVWGQGTLVTVSS BDBV407_VL (SEQ ID NO: 258)EIVMTQSPLSLSVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGADFTLKISRVEAEDVGVXYCMQA  BDBV425_VH(SEQ ID NO: 259) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFGISWVRQAPGQGLEWLGWINTYNGDTNYAQKFQGRVTMTFDTSTSTGFMELRSLRSDDFAVYYCARDSHLISIAVANTPNDFWGQGTLVTVSS BDBV425_VL (SEQ ID NO: 260)EIVMTQSPGTLSLSPGDRVTLSCRASQSVYSYYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQYYGNSHQGAA FGQGTKVEVK BDBV426_VH(SEQ ID NO: 261) QVQLQESGPGLVKPSQTLSLTCNVSGGSISSDDRYWSWIRQPPGKGLEWLGFIYYSGSTDYNPSLKSRVTMSLDTSKNQFSLKLNSVTAADTAMYYCATVTAYSPATMIVVGTEHGFDYWGQGTLVTVSS BDBV426_VL (SEQ ID NO: 262)DIVMTQSPSSLSASVGDRVTIFCRATQSIRSFLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQ GTKVEIK BDBV317_VH(SEQ ID NO: 263) QVQLVESGGGVVQPGGSLRLSCEVSGLTFSNFGMQWVRQAPGKGLEWVAFIRFDGSNKYYADSVKGRFTISRDNSKNTVYLQMGSLRAEDTAVYFCGRVL YGAAADFWGQGTLVTVSSBDBV317_VL (SEQ ID NO: 264)DIVMTQSPDSLAVSLGERATINCTSSHSLLYSSDNKNYLTWYQQKAGQPPKLLIYWASTRQSGVPDRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYTK SFTFGQGTKVEIKBDBV342_VH (SEQ ID NO: 265)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGKPNYAQKFQGRVTITADKSTSTAYMELRSLRSEDTAVYYCARGQGEIVVMVGHDDGGDYLGYWGQGTLVTVSS BDBV342_VL (SEQ ID NO: 266)QSALTQPRSVSGSPGXSVTISCTGTSSNVGAYNYVSWAQQMPGKAPKLMIFDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADFYCYSYAGSYTWI FGGGTKLTVLGBDBV357_VH (SEQ ID NO: 267)QVQLVQSGPGLVKPSETLSLTCSVSGGSISGSIWTWIRQSPGKGLEWIGYISLSGSTNFNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHRKSSKMVRGIEVFYYYYMDVWGKGTLVTVSS BDBV357_VL (SEQ ID NO: 268)QSALTQPASVSGSPGQSITISCTGTISDIGGYDYVSWYQQHPGKAPKLMIYDVSDRPSGVSNRFSGSKSGNTASLTISGLQSEDEADYYCSSYTRTYTPH VVFGGGTKLTVLGBDBV340_VH (SEQ ID NO: 269)QVQLVQSGPGLVKPSQTLSLTCTVSGGSISSGSFYWSWIRQPAGKGLEWIGRFYTTGSTHYNPSLKSRVTISADTSKNHFSLNLTSLTAADTAVYYCARGPVSYYSGNLYYFDYWGLGTLVTVSS BDBV340_VL (SEQ ID NO: 270)QSALTQPASVSGSPGQSITITCTGTSSDIGNNNYVSWYQQHPGKAPKLIIFDVNKRPSGVSNRFSGSKSDNTASLTISGLOAEDEADYYCSSYTNNRTFS FGGGTKVTVL BDBV392_VH(SEQ ID NO: 271) QVQLVQSGGGVVQPGGSLRLSGAASGFTFSSFGIHWVRQAPGKGLEWVAFIRYDGSDKFYLDSVKGRFTISRDNSKNTLFLQMSSLRVEDTAVYYCAKRGGHDYGYYDNNRYIDLWGRGTLVTVSS BDBV259_VH <Not Yet Available> BDBV259_VL(SEQ ID NO: 272) SYVLTQPPSVSVSPGQTASITCSGDKLGDRYTCWYQQKPGQSPVLVIYQDTKRPSGIPERFSGSNSGNTATLTISETQAIDEADYYCQAWDTS BDBV328_VH<Not Yet Available> BDBV328_VL (SEQ ID NO: 273)DIQMTQSPSTLSASVGDRVTITCRASQSISTYLAWYQQKPGKAPNLLIYIGASSLQSGVPPRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSYWWTFG QGTKVEII

TABLE 4 Variable Region Nucleic Acid Sequences BDBV43 VH (SEQ ID NO: 13)caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcagggcttctggagactccttcagccgcaagtatggcatcagctgggtgcgacaggcccctggacaaggatttgagtggatgggaacgatcatgccaatcgttggtttgaccacctccgcccagaaattccagggcagagtcacaattaccgcggacaagtccacgagcacagcccacatggaactgaacagcctgacatctgaggacacggccatttattactgtgcgagagatgaaattattggggctcgaccccactggttcgactcttggggccagggaaccctggtcacc gtctcctcaBDBV43 VL (SEQ ID NO: 14)gaaattgtgatgacccagtctccagccatcatgtctgtgtctccagggaaaagagccaccctctcctgcagggccagtcagagtgtcagtagcaacttagcctggtaccagcggaaacctggccaggctcccaggctcctcatctatggttcttccaccagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaggattttgcagtttattactgtctgcaatattataactggcctcggacgttcggccaagggaccaaggtggaaatcaaa BDBV223 VH (SEQ ID NO: 15)caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcacgactacctactggaattggatccgccagcccccagggaaggggctggaatggataggggaagtcaattatagtggaaacgccaactacaacccgtccctcaagggtcgagtcgccatatcagtggacacatccaagaaccagttctccctgaggttgaactctgtgaccgccgcggacacggctatatattactgtacgagtcgcatacgttcgcacattgcctactcgtggaagggggacgtctggggcaaagggaccacggtcaccgtc tcctcaBDBV223 VL (SEQ ID NO: 16)gaaattgtgatgacccagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttcccaggaattatataggttggttccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccgctggcttcccagacagattcagtggcagtgggtctgggacagacttcactctcaccatcaccagactggagcctgaagattttgcaatgtattactgtcaccagtatgataggttaccgtacacttttggccaggggaccaagctggagatcaaa EBOV49 VH (SEQ ID NO: 17)gaggtgcagctggtggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatgaaatcaactgggtccgccaggctccagggagggggctggagtgggtttcatacattagtagtagtggtagaaccatatactacgcagactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtttattactgtgcgagagaaccatatgttgacggaatattatatggggccggggatagctactttgactactggggccagggaaccctggtcaccgtctcctca EBOV49 VL (SEQ ID NO: 18)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaaggtcctgatctatgctgcatccagtttgcaaagtggggtctcatcaaggttcagtggcagtggatctgggacagacttcactctcaccatcagcagtctgcaacctgaagattttgcaacttacttctgtcaacagagttacaatacccctccggtcaccttcggccaagggacacgactggagattaaa EBOV52 VH (SEQ ID NO: 19)caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcaactgcactgtctctggtggctccatcagtagttactactggagctggatccggcagcccccagggaagggactggagtggattgggtatatctatgacagtgggagaaccaagtacaacccctccctcaagagtcgagtcaccatatcattagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagacacggccgtgtattactgtgcgagtctgggccctttcgacaaattatggttcggggagttgttgccgggatggttcgacccctggggccagggaaccctggtcaccgtctcctca EBOV52 VL (SEQ ID NO: 20)gaaattgtgttgacacagtctccagccaccctgtctttgtctccagggggaagagccaccctctcctgcagggccagtcagagtgttcacaactacttagcctggtaccaacagaagtctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctggagcctgacgattttgcagtttattactgtcagcaccgtagcaactggctcactttcggcggagggaccaaggtggagatcaaa EBOV63 VH (SEQ ID NO: 21)gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcggcctctggattcaccttaaatttctataacatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagtaattacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaggaagtcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagattttgtccagctattaattccgcaaagggacgagtggcagggtgtccacgactactacggtatggacgtctggggccaagggaccctggtcaccgtctcctca EBOV63 VL (SEQ ID NO: 22)gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccagggggaagagccaccctctcctgcagggccagtcagagtgttagcaacagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcattctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcactatggtagctctcagctcactttcggcggagggaccaaggtggagatcaaa EBOV82 VH (SEQ ID NO: 23)gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcagcctctggattcacttttactaacgcctggatgaactgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaacactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaagacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagggaaaagtgactgtagtggtggtaactgctacgtggttgactactggggccagggaaccctggtcaccgtctcctca EBOV82 VL <Not Yet Available> EBOV90 VH(SEQ ID NO: 24)gaggtgcagctggtggagtctgggggaggcttggtaaagcctgggggggcccttagactcagctgtgcagcctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaaatgatggtgggacagcagactacgctgcacccgtgaaaggcagattcagcatctcaagagatgattcaaaaaacacgctttatctgcaaatgaacagcctgaaaatcgaggacacagccgtgtattactgtatcacgtttttacgccccgaccactggggccagggaaccctggtcaccgtctcctca EBOV90 VL<Not Yet Available> EBOV9 VH (SEQ ID NO: 76)CAGGTGCAGCTGGTGCAGTCTGGGGTTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTAAGCTTGGTATAGCAAACTACGCACAGAACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTATTACTGAGTAGCAGGGATGCTTTTGATATCTGGGGCCAAGGGACCCTGGTCAGC GTGTCGTCAEBOV9 VL (SEQ ID NO: 77)QVQLVQSGVEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPKLGIANYAQNAQKFQGRVTITADKSTSTAYMELSRLRSEDTALYYCARVLLSSRDAFDIWGQGTLVT VSSEBOV109 VH (SEQ ID NO: 78)CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCTGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGAAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGACCAACAAAGGAACTAACGGTCGCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA EBOV109 VL (SEQ ID NO: 79)GACATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA EBOV119 VH (SEQ ID NO: 80)CAGGTGCAGCTGGTGCAGTCTGGGGTTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTAAGCTTGGTATAGCAAACTACGCACAGAACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGAGGACACGGCCCTGTATTACTGTGCGAGAGTATTACTGAGTAGCAGGGATGCTTTTGATATCTGGGGCCAAGGGACCCTGGTCACC EBOV119 VL<Not Yet Available> EBOV62 VH (SEQ ID NO: 283)gaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagtagctattggatgagctgggtccggcaggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgcgaaatactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtctattactgtgcgagagatggattactcgggatcagtgatttattataccccatatactactttgactactggggccagggaaccctggtcaccgtctcctca EBOV62 VL (SEQ ID NO: 284)gacattgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagaacattgggagctatttaaattggtatcagcagaaaccagggaaagcccctaacctcctgatgtatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcaccagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtatccctcgaacttttggccaggggacccagctggagattaaa EBOV87_VH (SEQ ID NO: 285)caggttcagctggtgcagtctggagctgaggtgaagaggcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccagctacgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcggtaacaatggtaacacaaactatgcacagaagctccagggcagactcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtttattactgtgcgagagatgccgatattgtcgtggtggtaggtgctacggggacctactactacggtatggacgtctggggccaagggaccctggtcaccgtctcctca EBOV87_VL (SEQ ID NO: 286)gacatcgtgatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattagtagctggttggcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtataaaagttctctgaggacgttcggccaggggacccagctggagattaaa EBOV157 VH (SEQ ID NO: 287)caggtgcagctggtgcagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaacaactacatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaattttttatagcgatggtaccacatacaacgcagactccgtgaagggcagattcaccatctccagagacaattccaagaacacgctgtatcttcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagacaagcaagtggctacgacgcctactacatggacgtctggggccagggaaccctggtcaccgtctcctca EBOV157 VL(SEQ ID NO: 288)gacatygtgatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattaatagctggttggcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatcaggcgtctactttagaaagaggggtcccatcaaggttcagcggcagtggagctgggacagaattcactctcaccattagcagcctgcagcctgatgattttgcaacttattactgccaacaatatagtagtttcccgctcactttcggcggagggaccaaggtggagctcaaa BDBV289 VH (SEQ ID NO: 289)caggtgcagctggtgcagtctggggctgaagtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggagccaccttcggcagcgatactgtcacctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctttttttggtgaagcaaactacgcacagaggtttcagggcagagtcacgataaccgcggacaagtccacgaacacagcctacatggaactgagcagcctgagatctgaggacacggccgtgtacttctgtgcgagacaaataaacgagatggctacatttggggagatacattattatacgtacatggatgtctggggccaagggaccctggtcaccgtctcctca BDBV289 VL <Not Yet Available> BDBV41 VH<Not Yet Available> BDBV41 VL (SEQ ID NO: 290)gaaattgtgatgacccagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattaccagcacctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaacagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatcatagctcacttttcggcggagggaccaaggtggagatcaaa  BDBV231 VH (SEQ ID NO: 291)caggtgcagctggtgcagtcgggcccaggtctggtgaagccttcggagaccctgtccctcacctgcactgtctctagtgactccatcaggagttactcctggagctggctccggcagcccccagggaagggcctggagtggattgggtttatctattacagtgggaacatcaattacaacccgtccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttgtccctgaacctgagctctgtgaccgctgcggacacggccgtgtattattgtgcgagagattggattacgatttttgggaggtacttcgatgtctggggccgtggcaccctggtcaccgtctcctca BDBV231 VL(SEQ ID NO: 292)gacatcgtgatgacccagtctccagactccctggctgtttctctgggcgagagggccaccatcaactgcaagtccagccagaatcttttatacagctccaacaataagaacttcttaacttggtaccaacacaaaccaggacagcctcctaagctgctcatttcctgggcatctactcgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcactttattactgtcagcaatattatactattcctccaacgttcggccaagggaccaaggtggaaatcaaa BDBV275_VH (SEQ ID NO: 293)caggtgcagctggtgcagtctgggggaggcttcgtacagccagggcggtccctgagactgtcctgtacagcctctggattcaactttggtgattatgttatgagctgggtccgccaggctccagggaaggggctggagtgggtaggtttcattaggggcaaaacttttggtgcgacaacagagtacgccgcgtctgtgaaaggcagatttaccatctcaagggatgattccaaaagcatcgcctacctgcaaattaaatccctgaaaaccgaggacacagccgtctactattgtactagaagggccaccagcacctggtacgaggactattggggccagggaaccctggtcaccgtctcc tcaBDBV275_VL (SEQ ID NO: 294)gacatcgtgatgacccagtctccggactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagagtgttttatacacccccaacaatcataattacttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactgggcatctgcccgggaacccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccataagcagcctgcaggctgaggatgtggcagtttattactgtcagcaatatcatattcctccgtacagttttggccaggggaccaagctggagatcaaa BDBV315_VH (SEQ ID NO: 295)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtgactccatcagcagtggtagttactactggagctggatccggcagcccgccgggaagggactggagtggattgggcgtatctataccagtgggagcaccaactacaatccctccctcaagagtcgagtcaccatttcagtagacacgtccaagaaccagttctccctgaacctgagctctgtgaccgccgcagacacggccgtgtattactgtgcgagagatccgattacgatttttggaggggttattttcggctggggaatggacgtctggggccaagggaccctggtcaccgtctcctca BDBV315_VL (SEQ ID NO: 296)gacattgtgatgacccagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagaccctcctgcatagtaatggatacaactatttgtattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatccggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcccgtcactttcggccctgggaccaaagtggatatcaaa BDBV329_VH (SEQ ID NO: 297)caggtccagctggtgcagtctggggctgaagtgaagaagcctgggtcctcagtgaaggtctcctgcaaggcttctggaggcaccttcgacacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggattatccctgttcttggtatagtagattatgcacagaagttccagggcagagtcacaattactgcggccaaattcacgaacatagcctacatggagctgagcagcctgagatctgaggacgcggccgtgtattactgtgcgagaggcctgcggagcctttctccccggggacaagagggacctactccagcgcccgggtggagaagggctcaataccactactactacatggacgtctggggcacagggaccctggtcaccgtctcctca BDBV329_VL(SEQ ID NO: 298)gaaattgtgatgacccagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagtagcaactatttagcctggtaccagcaaaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggccagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattattgtcagcagtatggtagttcacccggcactttcggcggagggaccaaggtggagatcaaa BDBV335_VH (SEQ ID NO: 299)caggtgcagctgcaggagtcgggcccaggactggtgaggccttcacagaccctgtccctcacctgcactgtgtctggtggctccatcaacagtgatagttactactggaactggatccggcagcccgccgggaagggactggagtggcttgggcgtgtctataccagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccaggtctccctgaggctgaactccgtgaccgccgcagacacgggcgtatattactgtgcgagagtggtttgggggagttatcgttcctaccactactcctacggtatggacgtctggggccaagggaccctggtcaccgtctcctca BDBV335_VL (SEQ ID NO: 300)gaaattgtgatgacccagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttggcagcagctacttagcctggtaccagcagagacctggccaggctcccaggctcctcttctatggtgcatcctacagggccactggcatcccagacaggttcagtgccagtgggtctggaacagacttcagtctcaccatcaacagactggagcctgaagattttgcagtctattactgtcagcagtctggtagctcgccggagacttttggccaggggaccaagctggagatcaaa BDBV354_VH (SEQ ID NO: 301)caggtgcagctggtgcagtctggagttgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcgtctggttacgcctttaccacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggttggatcagcacttactatggtaccacatactatgcacagaacctccagggcagagtcaccatgaccacagacacatccacgagcacatcctacttggaactgaggagcctaagatctgacgacacggccgtctattactgtgtgagagatcggtcgtggctggccacttcccgaccatatgatgcttttgatatctggggccaagggaccctggtcaccgtctcctca BDBV354_VL (SEQ ID NO: 302)gccatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggacattagcagtactttagcctggtatcagcagaaaccgggaaaagctcctaaactcctgatctatggtgcctccagtttggaaagtggggtcccatccaggttcaacggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatttcgcaacttattactgtcagcacttttactatttcccccgcaccttcggccaagggacacgactggagattaga BDBV386_VH (SEQ ID NO: 303)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtcgtttctactggagctgggtccggcagcccgccgggaggggactggagtggattgggcgcatctataccagtgggagcaccaactacaacccctccctcaagagtcgagtcagcatatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagatacggccgtgtattactgtgcgactgaactgtactactatggttcggggagttatgacccgctttggtcctggggccagggaaccctggtcaccgtctcctca BDBV386_VL (SEQ ID NO: 304)gacattgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcagggcattaacaacaatttaaattggcatcagcaaaaaccaggtaaagcccctaagctcctgatctacgatgcatccaatttggaaagaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgctacatattactgtcaacagaatgccaatctcccgcacacttttggccaggggaccaagctggagatcaaa BDBV397_VH (SEQ ID NO: 305)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtgggagttacttctggaactggatccggcagcccgccgggaagggactggagtggattgggcgtatctataccagcgggaccaccaactacaatccctccctcaagagtcgcctcaccatttcagtagacacgtccaagaaccaattctccctgaagctgaactctgtgaccgccgcagacacggccgtgtattactgtgcgacaagcccgtattactatgatagttctcattattacgactactggggccagggaaccctggtcacc gtctcctcaBDBV397_VL (SEQ ID NO: 306)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacattaccaactatttaaattggtatcagcagaagccagggaaagcccctaagctcctgatcttcgatgcttccaatttggaaaagggggtcccatcaaggttcagtgctactggatctgcgacagattttactttcaccatcagcagcctgcagcctgaagatactgcgacatattactgtcaacagtctgctgatctccccctcaccttcggccaagggacacgactggacattaaa BDBV399_VH (SEQ ID NO: 307)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcaatgtctctggtggctccatcagcaatggtggttaccactggagttggatccgccaggtcccagggaagggcctggagtggattggacacatttattacagtgggagcacctcctacaccccgtccctcaagagtcgacttaccatatcagtggacacctctaagaaccagttctccctgaagctgagctctgtgactgccgcggacacggccgtatattactgtgcgagagataggatacggggcgggcccattgactactggggccagggaaccctggtcaccgtctcctca BDBV399_VL(SEQ ID NO: 308)gacatccagatgacccagtctccatcctccctgtctgcatctgttggagaaagagtcaccatcacttgccgggcgagtcagggcatcgacaattatttagcctggtatcaacaaaaaccagggaaagttcctaaactcctgatctatgctgcatccactttgcactcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaggtataaccttgccccgagcgcttttggccaggggaccaaggtggagatcaga BDBV353_VH (SEQ ID NO: 309)caggtgcagctggtgcagtctggggctgagatgaggaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcagtgactactatatacactgggtgcgccaggcccctggacaagggcttgagtggctgggatggatcaacccttatagtggaggcacaaattatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcccacatggagctgagcgggctcagatctgacgacacggccctatatttctgtgcgagactatatggtgcggggagtcattataatcactacaacggcatggacgtctggggtcaagggaccctggtcaccgtctcctca BDBV353_VL (SEQ ID NO: 310)gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattggtagtttattggcctggtatcagcagaaaccggggaaagcccctaagctcctgatctatagggcgtctactttacaaggtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgatgttgctacttattactgccaacaatttaatagttatttcggcggagggaccaaggtggagatcaaa BDBV410_VH (SEQ ID NO: 311)caggtgcagctgcagcagtcgggcccaggactggtgaggccgtcacagaccctgtccctcacctgctctgtctctggtggctccgtcagtagtggtcgttacttctggaactggatccggcagtccgccgggaagggactggagtggattgggcgtatccattccagtgggagaaccaactccaacccctccctcaagagtcgagtcaccatatcagtcgacacgtccaagaaccagttctccctgcacctgggctctgtgaccgccgcagacacggccgtctattactgtgcgagaga BDBV410_VL(SEQ ID NO: 312)gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaagtctagtcagagcctcctgcatagtaatggagagacctatttattttggtacctgcagaagccaggccagccgccacaactcctgatctatgaagtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaagatcagccgggtggaggctgaggatgttggagtttattactgcatgcaaagtgtactccttccgtacacttttggccaggggaccaagctggagatcaag BDBV270_VH (SEQ ID NO: 313)caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtgcctccatcagcaggggtctttactactggagctggatccggcagcccgccgggaagggactggagtggattgggcgcatctataccagtgggagcatcaactacaatccttccctcaagagtcgagtcaccatatcagtagacacgtccaagaatcagttctccctgaggctgagctctgtaatcgccacagacacggccgtgtattattgtgtgagagatgctccctggggagattttttgactggttattttggcttctacggtatggacgtctggggccaagggaccctggtcaccgtctcctca BDBV270_VL (SEQ ID NO: 314)gacattgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattaacacctatttaaattggtatcagcagaaaccagggaaagcccctaagttcctgatctatgctgcatccagtttgcacagtggggtcccatcaaggttcagtggtagtggatctgggacagatttcactctcaccatcaacagtctacaacctgatgattttgcaacttactactgtcaacagagtttcactaccccgtacacttttggccaggggaccaagctggagatcaag BDBV324_VH (SEQ ID NO: 315)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgtaagacttctggatacaccttcaccagctttgaaatccactgggtgcgacagggcagtggacaagggcttgagtggatgggacgtatgaatcctaaaagtggtgacacagtctctgcacagaagttccagggcagagtcacccttaccagggacacgtccataaatgcagcctacatggagctgggcagcctgagttctgaggacacggccgtgtactactgtgcgagaggcccacacgttggcgaagttgttccaggtcttatggcgggcacctactattttcctttggacgtctggggccaagggaccctggtcaccgtctcctca BDBV324_VL (SEQ ID NO: 316)gacatccagatgacccagtctccctccaccctgtctgcatctataggagacagagtcaccatcacttgccgggccagtcagagcattagtcgctggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctataaggtgtctgatttacaaagtggggtcccatcaaggttcagcggcagtggatatgggacagaattcactctcaccatcggcagcctgcagcctgatgatttggcaacttattattgccaacaatatgatacatatccgtggacgttcggccaggggaccaagctggagatcaag BDBV403_VH (SEQ ID NO: 317)caggtccagctggtgcaatctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaacgcttctggaggcaccttcagcaactccattcttaactgggtgcgacaggcccctggacaagggcttgagtggatgggaaggatcatccctatcgttggtctagtaaacttcgcacaaaagttcgagggcagagtcacatttaccgcggacaaattcacgaacacagcctacatggagctgaacagtctgagatttgaggacacggccgtgtactactgtgcgataaatggggtaaatatcccggatactttgactactggggccagggaaccctggtcaccgtctcctca BDBV403_VL(SEQ ID NO: 318)gacattgtgatgacccagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgtgagcagcagctacttagcctggtaccagcaccaacctggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcatcatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggaagctcagcgatcaccttcggccaagggacacgactggagatcaag BDBV407_VH (SEQ ID NO: 319)caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcgctgtctctggtggctccatcaggagttatttctggagctggatccggcaggccccagggaagggactggaatggattgggaatatctattacagtgggcgccccaattacaacccctccctcaagaatcgagtcaccatatcagcagacacgtccaacaatgaggtctcactggagctgagcgctgtgaccgctgcggacacggccgtgtatttctgtgcgagagatgagagactactggtggaggtcggaaccgaccacttctactacggtttggacgtctggggccaagggaccctggtcaccgtctcctca BDBV407_VL (SEQ ID NO: 320)gaaattgtgatgacccagtctccactctccctgtctgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctacatagtaatggatacaactttttggattggtatttgcagaagccagggcagtctccacagctcctgatttatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatccggcgcagattttacactgaaaatcagcagagtggaggctgaggatgttggggtnnattactgcatgcaagct BDBV425_VH(SEQ ID NO: 321)caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcctcgggctacacctttaccagttttggtatcagctgggtgcgacaggccccgggacaagggctagagtggctgggatggatcaacacttacaatggtgacacaaactatgcacagaagttccagggcagagtcaccatgacaacagatacatccacgagtacaggcttcatggagctgaggagcctgagatctgacgacacggccgtctattactgtgcgagagactcccacttaataagtatagcagtggctaatacgcccaatgacttctggggccagggaaccctggtcaccgtctcctca BDBV425_VL (SEQ ID NO: 322)gaaattgtgatgacccagtcgccaggcaccctgtctttgtctccaggggacagagtcaccctctcctgcagggccagtcagagtgtttacagctactacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatgtatgatgcatccatcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagtctggagcctgaagattttgcagtgtactactgtcagtactatggtaactcacaccagggggcggcgttcggccaagggactaaggtggaagtcaaa BDBV426_VH (SEQ ID NO: 323)caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcaatgtctctggtggctccatcagcagtgatgatagatactggagctggatccgccagcccccagggaagggcctggagtggcttgggttcatctattacagtgggagcaccgactacaacccgtccctcaagagtcgagttaccatgtcactagacacctccaagaaccagttctccctgaagctgaactctgtgactgccgcagacacggccatgtattactgtgccacagtaacagcttactctcctgctactatgatagtagtgggtaccgaacatgggtttgactactggggccagggaaccctggtcaccgtctcctca BDBV426_VL (SEQ ID NO: 324)gacattgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcttttgccgggcaactcagagcattcgcagctttttaaattggtatcagcagaaaccagggaaagcccctaacctcctgatctatgctgcatccagtttgcaaagtggggtcccatccaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttactactgtcaacagagttacagtaccccatggacgttcggccaagggaccaaggtggagatcaag BDBV317_VH (SEQ ID NO: 325)aggtgcagctggtggagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtgaagtgtccggactcaccttcagtaactttggcatgcagtgggtccgccaggctccaggcaagggtctggagtgggtggcctttatacggtttgatggaagtaataagtattatgcagactccgtgaagggccgattcaccatatccagagacaactccaagaacacggtttatctccaaatgggcagcctgagagccgaggacacggcagtgtatttttgtgggagagttctatacggagccgcagctgacttttggggccagggaaccctggtcaccgtctcctca BDBV317_VL(SEQ ID NO: 326)gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcacgtccagtcacagtcttttatacagctccgacaataagaactatttaacttggtaccagcagaaagcaggacagcctcctaagctgctcatctactgggcttctacccggcaatccggggtccctgaccgattcagtggcagcgggtctgggacagagttcactctcaccatcagcagcctgcaggctgaagatgtggcagtctattactgtcagcagtattatactaagtctttcactttcggccaagggaccaaggtggagatcaag BDBV342_VH (SEQ ID NO: 327)caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcgtcggtgaaggtctcctgcaaggcctctggaggcaccttcagcagctatgctatcaactgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtaaaccaaactacgcacagaagttccagggcagagtcacgattaccgcggacaaatccacgagcacagcctacatggaactgagaagcctgagatctgaggacacggccgtatattactgtgcgcggggacagggagagattgtggtgatggttggtcatgacgacgggggggactaccttggctactggggccagggaaccctggtcaccgtctcctca BDBV342_VL (SEQ ID NO: 328)cagtctgccctgactcagcctcgctcggtgtccgggtctcctggacwgtcagtcaccatctcctgcactggaaccagcagtaatgttggtgcttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttttgatgtcactaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctggactccaggctgaggatgaggctgatttttactgctactcatatgcaggcagctacacttggattttcggcggagggaccaagctgaccgtcctaggt BDBV357_VH (SEQ ID NO: 329)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcagtgtctctggtggctccatcagtggttccatctggacctggatccggcagtccccagggaagggactggagtggattggatatatctctttaagtgggagcaccaacttcaacccctccctcaagagtcgagtcaccatttcagtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagacactgccgtgtattactgtgcgagacatcggaaatcgtcgaagatggttcgaggaattgaagttttctactactactacatggacgtctggggcaaagggaccctggtcaccgtctcctca BDBV357_VL (SEQ ID NO: 330)cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccatcagtgacattggtggttatgactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgatgtcagtgatcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccagtctgaggacgaggctgattattactgcagttcatatacaagaacttacactccccacgtggtattcggcggagggaccaagctgaccgtcctaggt BDBV340_VH (SEQ ID NO: 331)caggtgcagctggtgcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtggaagtttctactggagctggatccggcagcccgccgggaagggactggagtggattgggcgtttctataccactggaagcacccactacaatccctccctcaagagtcgagtcaccatatcggcggacacgtcgaagaaccacttctccctgaacctcacttctttgaccgccgcagacacggccgtttattactgtgcgagagggccggtctcctattatagtggcaacctctactactttgactactggggcctgggaaccctggtcaccgtctcctca BDBV340_VL (SEQ ID NO: 332)cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatcacctgcactggaaccagcagtgacattggtaataataactatgtctcctggtaccaacagcacccaggcaaggcccccaaactcatcatttttgatgtcaataagcgaccctcaggggtttctaaccgcttctctggctccaagtctgacaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaaacaacaggactttctccttcggaggtgggaccaaggtcaccgtccta BDBV392_VH (SEQ ID NO: 333)caggtgcagctggtgcagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctttggcatccactgggtccgccaggctccgggcaaggggctggagtgggtggcatttatacgatatgatggaagtgataagttctatttagactccgtgaagggccgattcaccatctctagagacaattccaagaatacgctgtttctgcaaatgagcagccttagagttgaagacacggctgtgtattactgtgcgaagagaggggggcatgattatggttactacgacaacaatcgctacatcgatctctggggccgtggcaccctggtcaccgtctcctca BDBV259_VL (SEQ ID NO: 334)tcctatgtgctgactcagccaccctcagtgtccgtgtccccgggacagacagccagcatcacctgctctggagataaattgggggatagatatacttgctggtatcaacagaagccaggccagtcccctgtattggtcatctatcaagatactaagcggccctcagggatccctgagcgattctctggctccaactctgggaacacagccactctgaccatcagcgagacccaggctatagatgaggctgactattactgtcaggcgtgggacaccagca BDBV415_VH (SEQ ID NO: 335)caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagttatggtgttagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctaagtttgctacagcaaaatacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgggacacttcccccagaggaaaccgattactacgatagtagtgattacttactggtccctcgatctctggggccgtggcaccctggtcaccgtctcctca BDBV343_VH (SEQ ID NO: 336)caggtgcagctggtggagtctggggctgaggggaagaagcctgggtcctcggtgaaggtctcctgcaaggctccaggagtcaccttcagcagatataccatcagctgggtgcgacaggcccctggacaggggcttgagtggatgggaaggatcagcccaatccttggcacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacaaatcctcgagcacagtctacatggaactgaacagactgaaatctgacgacacggctgtatattactgtgcgagagatgcaccgattattctggttgagggaccggagaccggtatggacgtctggggccaagggaccctggtcaccgtctcctca BDBV343_VL (SEQ ID NO: 337)cagtctgccctgactcagcctcgctcagtgtccgcgtctcctggacagtcagtcaccatctcctgcactggcaccaacagtgatgttggtggttatgactatgtctcctggtaccagcaacacccaggcaaagcccccaaactcatgatttctgatgtcaatatgcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaatctgaggatgaggctgattattactgctgctcatatgcaggcagctacacttttgtcttcggaagtgggaccaaggtcaccgtcctaggt BDBV377_VH (SEQ ID NO: 338)caggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcaatagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtttgatggaagtaaaaaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaactcactgtacttgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaaagacctcctgtatggttcggggatggtcccaaattactactactacggtttggacgtctggggccaagggaccctggtcaccgtctcctca BDBV377_VL <Not Yet Available> BDBV255_VH(SEQ ID NO: 339)caggtgcagctggtgcagtctgggggaggcctggtcaggcctggggggtccctgagactctcctgtacggcctctggattcaccctcagtacttatagcatgacctgggtccgccaggctccagggaagggcctggagtgggtctcatccatcagtagttcgtctacctacaagtactacgtggactcgattaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatggagagcctgggagtcgaggacacggctgtgtattactgttcgagagcggactgggactccgggaaaggagaccttgactcctggggccagggaaccctggtcaccgtctcc tcaBDBV255_VL (SEQ ID NO: 340)cagcctgtggtgactcagtcgccctctgcctctgcctccctgggagcctcggtcagactcacctgcactctcaacagcgggcgcagtaaatacgccatcgcatggcaccagcaacagccagggaagggccctcgctacttgatgacacttaatcatgatggcagtcacagcaagggagacgggatcccttttcgcttctcaggctccagctctgggactgagcgctacctcaccatctccagcctccagtctgaggatgaggctgactattactgtcagacttggggcaagggcatcgtggtattcggcggagggaccaagctgaccgtcctaggt BDBV432_VH (SEQ ID NO: 341)caggtgcagctggtgcagtcgggcccacgactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtgactccagcggtcgttactactggagctggatccggcagaccccagggaagggactagaatggattgggtatatctcttacactgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcttcagacatgtccaagagccacttctccctgaacttgacctctgtgaccgctgcggacacggccgtgtattattgtgcgagagggggatggaacctcctagtaagctactttgacttctggggcctgggaaccctggtcaccgtctcctca BDBV432_VL<Not Yet Available> BDBV91_VH (SEQ ID NO: 342)caggtgcagctggtgcagtctggggctgagttgaagccgcctggggcctcagtgaaggtctcctgcaagccttctggatacacgttcaccgactactatatacactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaaaagtggagaaacacactatgcacagaagtttcggggctgggtcaccttgaccagggacacgtccatcagcacaacctacatggacctgaccaggctgaaatctgacgacacggccgtgtatttctgtgcgagaggggatctagagactacgatcttcttctacaacgctgtggacgtctggggccaagggaccctggtcaccgtctcctca BDBV91_VL (SEQ ID NO: 343)gacatccagatgacccagtctccatcttccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaactgagagtattggcatctatttaaattggtatcagcggaaaccagggaaggcccctaacctcctgatctttgctacatccagtttgcagagtggggtcccgtcaaggttcagtggcagtggatctgggacagaattcactctcaccatcagcagtctgcaacctgaagattttgcaacttacttttgtcaacagggtttcagttctcctttcagttttggccaggggaccaggctggagatcaag

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,366,241-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,554,101-   U.S. Pat. No. 4,680,338-   U.S. Pat. No. 4,816,567-   U.S. Pat. No. 4,867,973-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 5,021,236-   U.S. Pat. No. 5,141,648-   U.S. Pat. No. 5,196,066-   U.S. Pat. No. 5,563,250-   U.S. Pat. No. 5,565,332-   U.S. Pat. No. 5,856,456-   U.S. Pat. No. 5,880,270-   “Antibodies: A Laboratory Manual,” Cold Spring Harbor Press, Cold    Spring Harbor, N.Y., 1988.-   Abbondanzo et al., Am. J. Pediatr. Hematol. Oncol., 12(4), 480-489,    1990.-   Allred et al., Arch. Surg., 125(1), 107-113, 1990.-   Atherton et al., Biol. of Reproduction, 32, 155-171, 1985.-   Brown et al., J. Immunol. Meth., 12; 130(1), 111-121, 1990.-   Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques    in Biochemistry and Molecular Biology, Vol. 13, Burden and Von    Knippenberg, Eds. pp. 75-83, Amsterdam, Elsevier, 1984.-   Capaldi et al., Biochem. Biophys. Res. Comm., 74(2):425-433, 1977.-   De Jager et al., Semin. Nucl. Med. 23(2), 165-179, 1993.-   Dholakia et al., J. Biol. Chem., 264, 20638-20642, 1989.-   Doolittle and Ben-Zeev, Methods Mol. Biol., 109, 215-237, 1999.-   Gefter et al., Somatic Cell Genet., 3:231-236, 1977.-   Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed.,    Orlando, Fla., Academic Press, 60-61, 65-66, 71-74, 1986.-   Gulbis and Galand, Hum. Pathol. 24(12), 1271-1285, 1993.-   Khatoon et al., Ann. of Neurology, 26, 210-219, 1989.-   King et al., J. Biol. Chem., 269, 10210-10218, 1989.-   Kohler and Milstein, Eur. J. Immunol., 6, 511-519, 1976.-   Kohler and Milstein, Nature, 256, 495-497, 1975.-   Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.-   Nakamura et al., In: Enzyme Immunoassays: Heterogeneous and    Homogeneous Systems, Chapter 27, 1987.-   O'Shannessy et al., J. Immun. Meth., 99, 153-161, 1987.-   Owens and Haley, J. Biol. Chem., 259, 14843-14848, 1987.-   Persic et al., Gene 187:1, 1997-   Potter and Haley, Meth. Enzymol., 91, 613-633, 1983.-   Remington's Pharmaceutical Sciences, 15th Ed., 3:624-652, 1990.-   Tang et al., J. Biol. Chem., 271:28324-28330, 1996.-   Wawrzynczak & Thorpe, In: Immunoconjugates, Antibody Conjugates In    Radioimaging And Therapy Of Cancer, Vogel (Ed.), NY, Oxford    University Press, 28, 1987.-   Beniac et al., (2012). The organisation of Ebola virus reveals a    capacity for extensive, modular polyploidy. PloS One 7, e29608.-   Brauburger et al., (2012). Forty-five years of Marburg virus    research. Viruses 4, 1878-1927.-   Brochet et al., (2008). IMGT/V-QUEST: the highly customized and    integrated system for IG and TR standardized V-J and V-D-J sequence    analysis. Nucleic Acids Res. 36, W503-508.-   Carette et al., (2011). Ebola virus entry requires the cholesterol    transporter Niemann-Pick C1. Nature 477, 340-343.-   Carragher et al., (2000). Leginon: An automated system for    acquisition of images from vitreous ice specimens. J. Struct. Biol.    132, 33-45.-   CDC (2009). Imported case of Marburg hemorrhagic    fever—Colorado, 2008. MMWR 58, 1377-1381.-   Chandran et al., (2005). Endosomal proteolysis of the Ebola virus    glycoprotein is necessary for infection. Science (New York, N.Y.)    308, 1643-1645.-   Cook, J. D., and Lee, J. E. (2013). The secret life of viral entry    glycoproteins: moonlighting in immune evasion. PLoS Path. 9,    e1003258.-   Côté et al., (2011). Small molecule inhibitors reveal Niemann-Pick    C1 is essential for Ebola virus infection. Nature 477, 344-348.-   Dias et al., (2011). A shared structural solution for neutralizing    ebolaviruses. Nat. Struct. Mol. Biol. 18, 1424-7.-   Dube et al., (2009). The primed ebolavirus glycoprotein    (19-kilodalton GP1,2): sequence and residues critical for host cell    binding. J. Virol. 83, 2883-2891.-   Dye et al., (2012). Postexposure antibody prophylaxis protects    nonhuman primates from filovirus disease. Proc. Natl. Acad. Sci    U.S.A. 109, 5034-5039.-   Garbutt et al., (2004). Properties of replication-competent    vesicular stomatitis virus vectors expressing glycoproteins of    filoviruses and arenaviruses. J. Virol. 78, 5458-5465.-   Giudicelli et al., (2011). IMGT/V-QUEST: IMGT standardized analysis    of the immunoglobulin (IG) and T cell receptor (TR) nucleotide    sequences. Cold Spring Harb. Protoc. 2011, 695-715.-   Hashiguchi et al., Cell 2015, in press.-   Johnson et al., (1996). Characterization of a new Marburg virus    isolated from a 1987 fatal case in Kenya. Arch. Virol. Suppl. 11,    101-114.-   Kajihara et al., (2012). Inhibition of Marburg virus budding by    nonneutralizing antibodies to the envelope glycoprotein. J. Virol.    86, 13467-13474.-   Ksiazek et al., (1999). ELISA for the detection of antibodies to    Ebola viruses. J. Infect. Dis. 179 Suppl 1, S192-198.-   Lander et al., (2009). Appion: an integrated, database-driven    pipeline to facilitate EM image processing. J. Struct. Bio. 166,    95-102.-   Lee et al., (2008). Structure of the Ebola virus glycoprotein bound    to an antibody from a human survivor. Nature 454, 177-182.-   Lubaki et al., (2013). The lack of maturation of Ebola    virus-infected dendritic cells results from the cooperative effect    of at least two viral domains. J. Virol. 87, 7471-7485.-   Maruyama et al., (1999). Ebola virus can be effectively neutralized    by antibody produced in natural human infection. J. Virol. 73,    6024-6030.-   Marzi et al., (2012). Protective efficacy of neutralizing monoclonal    antibodies in a nonhuman primate model of Ebola hemorrhagic fever.    PloS One 7, e36192.-   Murin et al., (2014). Structures of protective antibodies reveal    sites of vulnerability on Ebola virus. Proc. Natl. Acad. Sci. U.S.A.    111, 17182-17187.-   Nanbo et al., (2010). Ebolavirus is internalized into host cells via    macropinocytosis in a viral glycoprotein-dependent manner. PLoS    Pathog. 6, e1001121.-   Olinger et al., (2012). Delayed treatment of Ebola virus infection    with plant-derived monoclonal antibodies provides protection in    rhesus macaques. Proc. Natl. Acad. Sci U.S.A. 109, 18030-18035.-   Pettersen et al., (2004). UCSF Chimera—A visualization system for    exploratory research and analysis. J. Comput. Chem. 25, 1605-1612.-   Pettitt et al., (2103). Therapeutic intervention of Ebola virus    infection in rhesus macaques with the MB-003 monoclonal antibody    cocktail. Sci. Transl. Med. 5, 199ra113.-   Potter et al., (1999). Leginon: A system for fully automated    acquisition of 1000 electron micrographs a day. Ultramicroscopy 77,    153-161.-   Qiu et al., (2012). Successful treatment of Ebola virus-infected    cynomolgus macaques with monoclonal antibodies. Sci. Trans. Med. 4,    138ra181-138ra181.-   Qiu et al., (2014). Reversion of advanced Ebola virus disease in    nonhuman primates with ZMapp. Nature 514, 47-53.-   Saeed et al., (2010). Cellular entry of ebola virus involves uptake    by a macropinocytosis-like mechanism and subsequent trafficking    through early and late endosomes. PLoS Pathog. 6, e1001110.-   Saphire, E. O. (2013). An update on the use of antibodies against    the filoviruses. Immunotherapy 5, 1221-1233.-   Smith et al., (1982). Marburg-virus disease in Kenya. Lancet 1,    816-820.-   Suloway et al., (2005). Automated molecular microscopy: the new    Leginon system. J. Struct. Bio. 151, 41-60.-   Tang et al., (2007). EMAN2: an extensible image processing suite for    electron microscopy. J. Struct. Bio. 157, 38-46.-   Thomas et al., (1985). Mass and molecular composition of vesicular    stomatitis virus: a scanning transmission electron microscopy    analysis. J. Virol. 54, 598-607.-   Towner et al., (2009). Isolation of genetically diverse Marburg    viruses from Egyptian fruit bats. PLoS Path. 5, e1000536.-   Towner et al., (2006). Marburgvirus genomics and association with a    large hemorrhagic fever outbreak in Angola. J. Virol. 80, 6497-6516.-   Towner et al., (2005). Generation of eGFP expressing recombinant    Zaire ebolavirus for analysis of early pathogenesis events and    high-throughput antiviral drug screening. Virology 332, 20-27.-   van Heel et al., (1996). A new generation of the IMAGIC image    processing system. J. Struct. Bio. 116, 17-24.-   Warfield et al., (2007). Development of a model for marburgvirus    based on severe-combined immunodeficiency mice. Virol. J. 4, 108.-   Warfield et al., (2009). Development and characterization of a mouse    model for Marburg hemorrhagic fever. J. Virol. 83, 6404-6415.-   Warren et al., (2014). Protection against filovirus diseases by a    novel broad-spectrum nucleoside analogue BCX4430. Nature 508,    402-405.-   World Health Organization (2014a). Ebola Situation Report, in W.H.O.    Global Alert and Response. 7 Jan. 2015.-   World Health Organization (2014b). Marburg virus disease—Uganda, 10    Oct. 2014, in W.H.O. Global Alert and Response.-   Yu et al., (2008). An optimized electrofusion-based protocol for    generating virus-specific human monoclonal antibodies. J. Immunol.    Met. 336, 142-151.

1. A method of detecting an ebolavirus infection in a subjectcomprising: (a) contacting a sample from said subject with an antibodyor antibody fragment having clone-paired heavy and light chain CDRsequences from Table 2, respectively, or an antibody or antibodyfragment thereof as set forth in any figure or Table herein; and (b)detecting ebolavirus glycoprotein in said sample by binding of saidantibody or antibody fragment to antigen in said sample. 2-11.(canceled)
 12. A method of treating a subject infected with Ebolavirus,or reducing the likelihood of infection of a subject at risk ofcontracting Ebolavirus, comprising delivering to said subject anantibody or antibody fragment having clone-paired heavy and light chainCDR sequences from Table 2, respectively, or an antibody or antibodyfragment thereof as set forth in any figure or Table herein. 13-40.(canceled)
 41. A hybridoma or engineered cell encoding an antibody orantibody fragment, wherein the antibody or antibody fragment hasclone-paired heavy and light chain CDR sequences from Table 2,respectively, or is an antibody or antibody fragment thereof as setforth in any figure or Table herein.
 42. The hybridoma or engineeredcell of claim 41, wherein said antibody or antibody fragment is encodedby light and heavy chain variable sequences according to clone-pairedsequences from Table
 4. 43. The hybridoma or engineered cell of claim41, wherein said antibody or antibody fragment is encoded by light andheavy chain variable sequences having at least 70%, 80%, or 90% identityto clone-paired variable sequences from Table
 4. 44. The hybridoma orengineered cell of claim 41, wherein said antibody or antibody fragmentis encoded by light and heavy chain variable sequences having 95%identity to clone-paired variable sequences from Table
 4. 45. Thehybridoma or engineered cell of claim 41, wherein said antibody orantibody fragment comprises light and heavy chain variable sequencesaccording to clone-paired sequences from Table
 3. 46. The hybridoma orengineered cell of claim 41, wherein said antibody or antibody fragmentcomprises light and heavy chain variable sequences having 70%, 80%, 90%,or 95% identity to clone-paired sequences from Table
 3. 47. Thehybridoma or engineered cell of claim 41, wherein the antibody fragmentis a recombinant ScFv (single chain fragment variable) antibody, Fabfragment, F(ab′)₂ fragment, or Fv fragment, or incorporated as scFv orFab in a diabody.
 48. The hybridoma or engineered cell of claim 41,wherein said antibody is a chimeric antibody or is an IgG. 49.(canceled)
 50. The hybridoma or engineered cell of claim 41, whereinsaid antibody or antibody fragment further comprises a cell penetratingpeptide or is an intrabody. 51-69. (canceled)
 70. The method of claim12, the antibody is characterized by clone-paired variable sequences asset forth in Table
 3. 71. The method of claim 12, the antibody isencoded by clone-paired light and heavy chain variable sequences as setforth in Table
 4. 72. The method of claim 12, wherein said antibody orantibody fragment is encoded by light and heavy chain variable sequenceshaving 70%, 80%, or 90% identity to clone-paired sequences from Table 4.73. The method of claim 12, wherein said antibody or antibody fragmentcomprises light and heavy chain variable sequences having 70%, 80%, or90% identity to clone-paired variable sequences as set forth in Table 3.74. The method of claim 12, wherein the antibody fragment is arecombinant ScFv (single chain fragment variable) antibody, Fabfragment, F(ab′)₂ fragment, or Fv fragment, or incorporated as scFv orFab in a diabody.
 75. The method of claim 12, wherein said antibody isan IgG or is a chimeric antibody.
 76. The method of claim 12, whereinsaid antibody is administered prior to infection.
 77. The method ofclaim 12, wherein said antibody is administered after infection.
 78. Themethod of claim 12, wherein delivering comprises antibody or antibodyfragment administration, or genetic delivery with an RNA or DNA sequenceor vector encoding the antibody or antibody fragment.